WO2007002285A2 - Methods for treating neurological and psychiatric conditions - Google Patents
Methods for treating neurological and psychiatric conditions Download PDFInfo
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- WO2007002285A2 WO2007002285A2 PCT/US2006/024303 US2006024303W WO2007002285A2 WO 2007002285 A2 WO2007002285 A2 WO 2007002285A2 US 2006024303 W US2006024303 W US 2006024303W WO 2007002285 A2 WO2007002285 A2 WO 2007002285A2
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- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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- the invention relates generally to the field of neuropharmacology. More specifically, the invention relates to methods to treat or prevent neurological or psychiatric conditions mediated by astrocytes.
- the structure of astrocytes provided the first clues to their function in the nervous system.
- the processes of the astrocyte are contained within a distance of about lOO ⁇ m and processes of the same cell contact neuronal membranes and the vasculature, where they form endfeet around the endothelia and smooth muscle.
- the astrocyte makes contact with synapses in several regions of the brain in a structure that has been defined as a tripartite synapse: the astrocytic process is associated with the pre and postsynaptic elements of the synapse.
- the astrocyte can contact the vasculature and synapses, it has been proposed that one function of this cell is to provide metabolic support to the neuron, a function , that is probably important in supporting neuronal function during elevated levels of neuronal activity.
- this structural relation has led to the idea of a reciprocal arrangement in which activity in the synapse is transmitted through signaling cascades of the astrocyte to the vasculature to locally regulate blood flow to provide metabolic support tuned to the demand of the level of neuronal activity.
- synaptic level it has been estimated that the processes of one astrocyte make contact with over 100,000 synapses. This synaptic relation serves many functions which include the clearance of synaptic transmitters.
- a synaptic transmitter is released from the presynaptic nerve terminal to act on postsynaptic receptors, it is important that it is quickly cleared from the synapse in order to prevent receptor desensitization to maintain the fidelity of synaptic transmission.
- astrocytes Upon activation, astrocytes release chemical transmitters. The release of these gliotransmitters, in a process termed gliotransmission, can lead to paracrine actions on astrocytes supporting inter-astrocytic Ca signals, as well as signaling to neurons to regulate neuronal excitability and synaptic transmission. Astrocytes have been shown to release several transmitters, including glutamate, D-serine, ATP, adenosine, homocysteic acid, taurine and peptides such as atrial natriuretic factor (ANF).
- ANF atrial natriuretic factor
- Glutamate can have presynaptic effects mediated by either metabotropic glutamate receptors (mGluRs) or by kainite receptors that induce an enhancement of transmitter release.
- D-serine regulates NMDA receptor functions and synaptic plasticity.
- ATP can act through postsynaptic P2X receptors to induce an elevation of postsynaptic Ca +2 which is thought to drive the insertion of AMPA receptors to mediate an increase synaptic transmission.
- released ATP can cause a reduction in synaptic transmission mediated by presynaptic adenosine Al receptors, and modify neuronal excitability through the activation of GIRK (or Ikir) which hyperpolarizes the neuronal membrane potential.
- astrocyte-dependent regulation of synapses is mediated through adenosine, and that this gliotransmitter allows the astrocyte to act as an intermediary in signaling between networks of synapses. It is well known that there is a tonic level of extracellular adenosine in the brain which causes a presynaptic inhibition of transmitter release from certain excitatory synapses. Additionally, adenosine can accumulate in an activity- dependent manner.
- GFAP glial fibrillary acidic protein
- TLE Temporal lobe epilepsy
- Status epilepticus induces a variety of alterations in the nervous system occurring at distinct periods following the onset of status epilepticus.
- GABA receptors are internalized. Later, after the termination of status epilepticus, during a latent period in which behaviorally detected seizures are not occurring, the so-called latent period of epileptogenesis, new GABA receptors with a different sub-unit composition are expressed ( Otis TS et al. (1994) Proc. Natl. Acad. Sci. USA 91:7698-7702; Brooks-Kayal AR et al. (1998) Nat. Med. 4:1166-72; Nusser Z et ⁇ l.
- epilepsy epileptogenesis
- the current therapeutic strategy is to treat epileptic patients with anticonvulsants. There is currently no treatment following the initial insult that will prevent epileptogenesis.
- seizure disorders One percent of the population has a seizure disorder and ten percent of the population will have a seizure during its lifetime. In some patients, seizure symptoms can be alleviated or reduced in frequency by the lifelong use of anti-convulsants.
- relatively little headway has been made in understanding the mechanisms that lead to the generation of epilepsy following an episode of status epilepticus or after other types of injury to the nervous system. For example, following a traumatic head injury, there is no known treatment that will prevent epileptogenesis even though we know that up to 50% of individuals with moderate to severe traumatic brain injury are highly likely to develop epilepsy.
- astrocytes include frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (FormanMS et al. (2005) J. Neurosci. 25:3539-50).
- Reactive astrocytosis can also play a supportive role following neuronal injury.
- ablation of astrocytes in mice with spinal cord injuries demonstrated failure of the blood brain barrier, demyelination, neuronal death, and motor impairment compared with parallel injured mice in which astrocytes were maintained (Faulkner JR et al. (2004) J. Neurosci. 24:2143-55).
- ALS amyotrophic lateral sclerosis
- Gliotransmission may play a role in schizophrenia.
- schizophrenia is a complex disorder in which abnormalities in dopamine signaling have been strongly implicated
- the recent discovery of the importance of NMDA receptor function is a breakthrough discovery.
- PCP phenycyclidine
- NMDA receptor antagonist an NMDA receptor antagonist
- PCP administration in rodents results in effects reminiscent of human schizophrenia such as working memory deficits, motor problems, and social behavior abnormalities.
- the invention features methods for treating or preventing neurological or psychiatric conditions in a subject.
- Various conditions can be treated or prevented using the inventive methods, and these conditions include, without limitation epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP-17, Hepatic encephalopathy, or Lupus encephalitis.
- ADHD attention deficit hyperactivity disorder
- the methods comprise modulating the expression or activity of one or more proteins that participate in calcium signaling within astrocytes.
- proteins include, but are not limited to mGluR receptors, Gq, phospholipase C, Regulator of G Protein Signaling (RGS), IP3 phosphatase, IP3 Receptor, P2Y1, P2Y2, P2Y4, PIP2 kinase, PIP2 phosphatase, and caibmdm.
- Calcium signaling is targeted to modulate gliotransmission oi various chemical transmitters such as, but not limited to, glutamate, adenosine, ATP, and D- serine.
- Modulating the expression or activity of the various proteins that participate in calcium signaling can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins.
- Various agonists and antagonists can be used to modulate the expression or activity of the proteins.
- Exemplary antagonists include, without limitation, MPEP, MTEP, SIB-1893. SEB-1757, GPant-2A, PPADS, and U-73122.
- the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of glutamate.
- proteins are SNARE proteins, synaptotagmin IV, Munc-18, VGLUTl, VGLUT2, or VGLUT3.
- Modulating the expression or activity of the various proteins that participate in the expression or release of glutamate can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins.
- Clostridial toxins and antagonists are exemplary ways to modulate the proteins that participate in the expression or release of glutamate.
- the methods comprise modulating the expression or activity of at least one protein that is activated by glutamate.
- proteins include NMDA receptors and AMPA receptors, and preferred targets include the NR2B subunit of the NMDA receptors.
- Modulating the expression or activity of the various proteins that are activated by glutamate can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins.
- Non-limiting examples of suitable antagonists are is D- AP5, (R)-AP5, PBPD, EAB515, LY233536, MK-801, Memantine, Ketamine, L-701,324, L- 689,560, GV196771A, Ro 25-6981, Co 101949, and Ifenprodil.
- the methods comprise modulating the oscillation of free cellular calcium, particularly in astrocytes.
- One preferred means to modulate the oscillation of free cellular calcium is administration of a calcium chelator.
- Non-limiting examples of calcium chelators suitable for use in the present invention include EDTA, EGTA, diazo-2, FURA-2, Di- Bromo-BAPTA, and BAPTA-AM.
- the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of ATP or adenosine.
- ⁇ " iu is ./ " ilir ⁇ -iii ci) ⁇ iir. 1 HT-. d; rut. r ⁇ , ⁇ . T A T , p , . , , « . . , TM ⁇ , r limiting examples of such proteins include SNARE proteins, bradyl ⁇ nin receptors, mGluR5,
- Modulating the expression or activity of the various proteins that participate in the expression or release of ATP or adenosine can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins.
- Clostridial toxins and agonists are exemplary ways to modulate the proteins that participate in the expression or release of ATP or adenosine.
- Exemplary agonists include, but are not limited to, OAG, DAG lipase inhibitor RHC-80267, glutamate, ATP, norepinephrine, acetylcholine, and bradykinin. Modulation can also be by means of inhibiting diacylglycerol kinase or diacylglycerol lipase.
- the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of D-serine.
- One non-limiting example of such proteins is the NMDA receptor.
- Modulating the expression or activity of the various proteins that participate in the expression or release of D-serine can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins.
- Exemplary agonists suitable for use in the present invention include, but are not limited to, ACBD, homoquinolinic acid, D-serine, cycloserine, D-cycloserine, glutamate, ATP, norepinephrine, acetylcholine, and bradykinin.
- Figure 1 shows cell-wide Ca +2 oscillations in astrocytes lead to elevated glial glutamate release, excitation of neurons and delayed neuronal death.
- Cell- wide Ca +2 elevations evoke the release of large quantities of glutamate that synchronously depolarize groups of pyramidal neurons (B3). This enhancement of gliotransmission stimulates delayed neuronal death and re-organization underling epileptogenesis.
- FIG. 2 shows that application of Fluo-4AM to the surface of the cortex leads to the loading of astrocytes with the Ca +2 indicator fluo-4 which is confirmed by co-loading with sulforhodarnine 101. Images were acquired -lOOum beneath the cortical surface in vivo.
- Figure 3 snows that injection into cortex with ⁇ uo-4AM (green) predominantly loads neurons, with weak labeling of astrocytes. However, subsequent surface labeling with SRlOl allows bright labeling of astrocytes (red).
- Figure 4 shows two photon photolysis and imaging in vivo. Caged nitric oxide was photoreleased onto cortical astrocytes labeled with the Ca 2 indicator fiuo-4 (pseudocolor display). Images shown before (left) following (middle, right) photolysis (right; scale 20um).
- Figure 5 shows that status epilepticus evokes a persistent increase in astrocytic Ca +2 signals lasting for three days.
- E Time course of change in Ca +2 oscillations reported as the integral of the ⁇ F/Fo signal (p ⁇ 0.002; n value is between 4 and 8 animals for each time point).
- FIG. 6 shows cell-wide and local astrocytic calcium oscillations in vivo.
- A) Astrocytes from mice 3 days after SE exhibit cell-wide Ca +2 oscillations.
- A) An example of astrocyte (green cell labeled with Fluo-4) showing cell-wide oscillations invaded from process 1 (Al) and process 2 (A2) to cell body.
- B) An example of astrocyte from a control mouse showing local oscillations of process 3, which did not invade to cell body.
- C-D Calcium oscillations of astrocyte shown in (A). Oscillations in cell body and processes are highly synchronized as shown in the time course of ⁇ F/F 0 . Boxes Al and A2 correspond to the images Al and A2 in A).
- E) Comparison of cross correlation coefficients at zero lag time in the absence and in the presence CHPG showing that this niGluR5 agonist stimulates coordinated Ca signals in groups of astrocytes (N 5-6 animals per group). * p ⁇ 0.05; ** ⁇ 0.02; ***p ⁇ 0.01.
- FIG. 8 shows astrocytic Ca +2 oscillation in vivo in mice three days post-SE is inhibited by mGluR5 antagonist MPEP.
- the NR2B NMDA receptor antagonist ifenprodil (20 ⁇ M) does not affect astrocytic Ca +2 signals.
- * p ⁇ 0.002 (N 3 to 4 animals in each group).
- Figure 9 shows that gliotransmission but not intra-cortical synaptic transmission activates the NR2B-containing NMDA receptors of layer 2-3 pyramidal neurons.
- A) Whole-cell recording from a pyramidal neuron showing SICs induced by bath application of CHPG (0.5 mM). In this as well as in panels B-E, experiments are performed in the continuous presence of TTX (1 ⁇ M).
- CHPG 0.5-1 mM or low Ca +2 containing ACSF.
- D-E Mean amplitude of SICs under control conditions, in the presence of ifenprodil (D, 3 ⁇ M) or NVP-AAM077 (E, 0.4 ⁇ M) and after drug washout. Data are normalized to the amplitude of SICs recorded under control conditions. See inset for representative SICs under the different experimental conditions from the same cell. Number of averaged SICs is 37, 48 and 70 from 9 cells for panel D and 27, 25, 13 from 7 cells for panel E respectively.
- FIG. 10 shows MPEP and ifenprodil protect neurons from status epilepticus- evoked death.
- C Time course of FJB labeling.
- D The region identified by the dashed lines in (B) is shown from animals treated with glutamate receptor antagonists, as labeled.
- Figure 11 shows photolytic elevation of Ca +2 in an astrocyte-evokes a slow inward current in CAl pyramidal neurons once the Ca +2 elevation in this glial cell confronts the neuronal dendrite (white overlay).
- D-AP5 blocks this glial induced neuronal current.
- the NR2B-selective antagonist ifenprodil was applied to slices and showed a selective reduction of SIC amplitude with little effect on synaptic NMDA receptors (Fellin et al, 2004). This demonstrates that glial glutamate acts on extrasynaptic NMDA receptors. Additionally, activation of NR2B-containing NMDA receptors can activate a neuronal cell death pathway.
- Figure 12 snows injection of adenovirus containing GFAP promoter into rodent cortex directs the expression of receptor-EGFP fusion construct in astrocytes (individual cells not by arrows) Calibration 50 ⁇ m.
- Figure 13 shows astrocyte-specific expression of the dn-SNARE domain.
- A Two lines of animals were generated, hGFAP.tTA and tetO.SNARE. When these lines are crossed, Dox suppresses SNARE, EGFP, and lacZ expression.
- B ⁇ -galactosidase ( ⁇ -Gal) expression on a parasagittal section of dn-SNARE mouse brain (scale bar, 1 mm).
- C and D Slices from dn-SNARE mice ( ⁇ Dox) show transgene regulation by Dox as reported by ⁇ -Gal (scale bar, 200 ⁇ m).
- E EGFP and ⁇ -Gal fluorescence and (F) GFAP immunoreactivity demonstrate expression of gene products in astrocytes.
- G Merged (E) and (F) images.
- H to J In hippocampal CAl, (H) EGFP and ⁇ -Gal transgenes are not expressed in neurons identified by (I) NeuN immunoreactivity.
- J Merged (H) and (I) images.
- Figure 14 shows astrocytes regulate synaptic transmission and modulate plasticity through the control of extracellular adenosine.
- Stim the dashed line represents the stimulus voltage (Stim) used to evoke the individual example traces shown as inserts.
- Figure 15 shows dn-SNARE expression in astrocytes reduces extracellular ATP, a source of adenosine that regulates synaptic transmission.
- Figure 16 shows dn-SNARE expression in astrocytes prevents adenosine- mediated heterosynaptic depression.
- A Schematic representation of the experimental setup, showing two independent pathways Sl and S2. Stimulation of pathway Sl, 50 ms before S2 (top trace), does not change the amplitude of S2 fEPSP compared to stimulation of S2 alone (middle trace). The lower trace shows the top and middle traces superimposed.
- DPCPX 800 nM
- Data are presented as mean ⁇ SEM.
- Figure 17 shows that using vasculature as landmarks (A, B), which is loaded with rhodamine dextran, individual astrocytes can be identified, after removal of the animal from the imaging platform and after reattachment, the same identified astrocytes can be identified for subsequent studies. This approach can be used to study the impact of pharmacological antagonists/agonists on cell survival.
- Figure 18 shows that transgenic mice which selectively express a dnSNARE transgene only in astrocytes (as defined in figures 13-16) to reduce the astrocyte-dependent accumulation of adenosine show a higher frequency of seizures in epileptic animals. Data are shown for three sequential analysis periods, in the presence and absence of doxycycline.
- Figure 19 shows that treatment of the cortex with B APTA/AM, which selectively chelates Ca +2 in astrocytes, attenuates neuronal death as assayed by fluorojade B labeling. P ⁇ 0.05
- epileptogenesis refers to any molecular or cellular event that is part of the inception, development, transition, or progression of epilepsy.
- the term "nervous system” refers to the entire nerve apparatus of an organism, and includes the central nervous system which comprises the brain and spinal cord, and the peripheral nervous system, which comprises the cranial and spinal nerves, autonomic ganglia, and plexuses, as well as the sympathetic and parasympathetic nervous systems.
- a “neuron” or “neuronal cell” refers to any cell in the nervous system that conducts nerve impulses.
- a condition is any state of being. This term is not limited to diseases or disorders, which can be considered an unhealthy state of being.
- a condition can be a transitional state of being.
- a condition can be a healthy or normal state of being.
- a subject having a condition need not manifest clinical symptoms.
- Psychiatric condition refers to conditions of the mind.
- Neurological condition refers to conditions of the nervous system.
- a "convulsion” is an involuntary contraction or series of contractions of the voluntary muscles. Convulsions can, but need not accompany a seizure.
- antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a protein.
- agonist is used in the broadest sense, and includes any molecule that enhances, stimulates, or facilitates a biological activity of a protein.
- Test compound refers to any purified molecule, substantially purified molecule, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material that can be analyzed using the methods of the present invention.
- Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof.
- Biomolecules include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof.
- Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized de novo.
- Test compounds can be defined in terms of structure or composition, or can be undefined.
- the compound can be an isolated product of unknown structure, a mixture of several known products, or an undefined composition comprising one or more compounds.
- undefined compositions include cell and tissue extracts, growth »'" ft perennial ft .- >- V ⁇ :;: ⁇ !' »... ⁇ .. » " , . ⁇ .. medium m which prokaryotic, eukaryotic, and archaebactenal cells have been cultured, fermentation broths, protein expression libraries, and the like.
- treating refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
- the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluations.
- the term "effective amount” refers to an amount of a biologically active compound or fragment, homolog, analog, or conjugate thereof sufficient to exhibit a detectable therapeutic effect.
- the therapeutic effect may include, for example, without limitation, the amelioration of a neurological or psychiatric condition.
- modulate refers to any change, including without limitation, any increase, decrease, enhancement, inhibition, suppression, or alteration, particularly with respect to the expression or activity of the proteins described herein, as well as the genes encoding said proteins.
- calcium oscillation refers to any fluctuation in calcium concentration, especially as occurs in cellular signal transduction.
- the invention provides methods for treating or preventing a neurological or psychiatric condition in a subject in need of such treatment. Such methods comprise modulating the expression or activity of one or more proteins that participate in calcium signaling within astrocytes.
- the methods are effective for treating or preventing a neurological or psychiatric condition in which the calcium signaling mediates the expression or release of a chemical transmitter by the astrocyte and/or wherein the expression or release of the transmitter mediates, in whole or in part, the neurological or psychiatric condition in the subject.
- inventive methods are not limited to conditions that are initiated, mediated, exacerbated, or sustained by the astrocyte itself. Because of the privileged access of astrocytes to neurons and their synapses, it is possible to change synaptic transmission and neuronal . , .. e- t . . _. , i • , j . • ⁇ i - ⁇ excitability by modulating gliotransmission.
- targeting molecules to astrocytes will enable gliotransmission to act through the neuron and synapse to ameliorate the condition. That is, targeting calcium signaling in or gliotransmission of any of the various chemical transmitters expressed or released by the astrocyte can provide a therapeutic or preventative effect for conditions that are initiated, mediated, exacerbated, or sustained by the synapse, neuron, or any other cell in which gliotransmission by an astrocyte will produce an effect.
- An astrocyte is a type of cell found in the brain and spinal cord.
- An astrocyte is a small, star-shaped glial cell (a cell that surrounds and supports nerve cells). The process by which astrocytes release chemical transmitters is referred to as gliotransmission.
- Ca +2 signals in astrocytes evoke the release of glutamate and astrocytic glutamate acts on neuronal NR2B-containing NMDA receptors, which have been associated with cell death.
- Neuronal death is a pathology observed in multiple neurological tissue diseases. Such diseases include epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Huntington's disease, Parkinson's disease, and peripheral neuropathy, to name only a few.
- the methods of the invention have application to prolong neuronal cell survival in such neurodegenerative diseases.
- the methods of the present invention are applicable to prolong neuronal cell survival in a subject that has suffered an insult, injury, trauma, or the like to the nervous system, especially an insult, injury, trauma, or the like to the brain.
- the injury to the nervous system may be a physical injury, particularly traumatic injuries, stroke, status epilepticus and the like.
- the survival of the neuronal cells is prolonged where cell death would be expected to occur, for example, at the inception, development, transition, or progression of neurodegenerative diseases.
- On preferred aspect of the invention utilizes methods to prolong neuronal cell survival for the treatment or prevention of epilepsy.
- proteins that participate in calcium signaling in the astrocyte include, without limitation, mGluR receptors such as niGluR5, GTP binding protein Gq, Regulator of G Protein Signaling (RGS) proteins, such as RGS2, IP 3 phosphatase, IP 3 receptors, P2Y receptors such as P2Y4, PIP2 kinase, PIP2 phosphatase, and calbindin.
- RGS Regulator of G Protein Signaling
- RGS2 proteins such as RGS2
- IP 3 phosphatase IP 3 receptors
- P2Y receptors such as P2Y4, PIP2 kinase, PIP2 phosphatase, and calbindin.
- the modulation of the production of such proteins can be effectuated, for example, by increasing or decreasing expression of one or more of the proteins at the genetic level such as by increasing or decreasing the expression of the genes that encode such proteins.
- Regulation of genes can occur, for example, at the transcription level, e.g., by inhibiting transcription. Regulation of genes can also occur, for example, at the post-transcription level, e.g., by inhibiting or degrading the mRNA.
- Antisense nucleic acids are one well-known approach to post-transcriptionally regulate gene expression. Full-length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be utilized. The use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art.
- RNA interference can also be used for post-transcriptional regulation of the proteins that participate in calcium signaling.
- RNAi RNA interference
- traditional methods of gene suppression employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the corresponding protein.
- RNAi also operates on a post-translational level and is sequence specific, but suppresses gene expression far more efficiently.
- Exemplary methods for controlling or modifying gene expression are provided in WO 99/49029, WO 99/53050 and WOO/75164, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
- post-transcriptional gene silencing is brought about by a sequence-specific RNA degradation process which results in the rapid degradation of transcripts of sequence-related genes.
- double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998).
- Gene constructs that produce transcripts with self- complementary regions are particularly efficient at gene silencing.
- RNAi specifically bind to and cleave double-stranded RNA into short fragments.
- the ribonuclease(s) remains associated with these fragments, which in turn specifically bind to complementary mRNA, i.e., specifically bind to the transcribed mRNA strand for the gene of interest.
- the mRNA for the gene is also degraded by the ribonuclease(s) into short fragments, thereby obviating translation and expression of the gene.
- an RNA-polymerase may act to facilitate the synthesis of numerous copies of the short fragments, which exponentially increases the efficiency of the system.
- silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism.
- siRNA short hairpin RNAs
- a vector containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell by any means suitable in the art. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to siRNA molecules and are used by the cell to mediate RNA silencing of the desired protein.
- nucleotide sequence, polypeptide sequence, etc. of the proteins that participate in calcium signaling or gliotransmission in the astrocyte can be used to generate gene silencing constructs and/or gene- specific self-complementary, double-stranded RNA sequences that can be delivered by conventional art-known methods.
- sequences for the various proteins described herein are known and can be readily obtained through any of the various databases known and available to those of skill in the art.
- a gene construct can be employed to express the self-complementary RNA sequences.
- cells can be contacted with gene-specific double-stranded RNA molecules, such that the RNA molecules are internalized into the cell cytoplasm to exert a gene silencing effect.
- the double-stranded RNA should have sufficient homology to the targeted gene to mediate RNAi without affecting expression of non-target genes.
- the double-stranded DNA is at least 20 nucleotides in length, and is preferably 21-23 nucleotides in length.
- the double-stranded RNA corresponds specifically to a polynucleotide of the present invention.
- siRNA small interfering RNA
- Methods of the present invention include the use of gene therapy to regulate RNA interference and to over-express proteins.
- expression can be selective to neurological tissue, and more specifically, selective to astrocytes through the use of astrocyte-specific promoters. See, e.g., Wang CY et al (2006) Gene Ther. 2006 May 25; [Epub ahead of print]; Namikawa K et al (2006) Gene Ther. 2006 Apr 20; [Epub ahead of print]; and, Vandier D et al (2000) Cancer Gene Ther. 7:1120-6.
- Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
- Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143- 4146 (1986)). The oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups.
- nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
- the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, ex vivo, or in vivo in the cells of the intended host.
- Techniques suitable for the transfer of nucleic acid into cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
- the currently preferred in vivo gene transfer techniques include transfection with viral vectors and viral coat protein liposome mediated transfection (Dzau et al, 1993, Trends in Biotechnology, 11 :205-210).
- Viral vector mediated techniques may employ a variety of viruses in the construction of the construct for delivering the gene of interest.
- the type of viral vector used is dependent on a number of factors including immunogenicity and tissue tropism.
- Some non- limiting examples oi viral vectors useful in gene therapy include retroviral vectors (see e.g., U.S. Patents 6,312,682, 6,235,522, 5,672,510 and 5,952,225, ), adenoviral (Ad) vectors (see e.g., U.S. Patents 6,482,616, 5,846,945 ), baculovirus vectors, and adeno-associated virus (AAV) vectors (see, e.g., U.S.
- Patents 6,566,119, 6,392,858, 6,468,524 and WO 99/61601 it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, and the like.
- an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, and the like.
- proteins which bind to a cell surface membrane protein associated with endocytosis can be used for targeting and/or to facilitate uptake, e.g.,. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
- calcium signaling facilitates enzymatic pathways that proceed to induce the expression and/or release of various chemical transmitters by the astrocyte.
- chemical transmitters produced by astrocytes include glutamate, D- serine, and ATP, which is converted in the extracellular space to adenosine.
- the invention contemplates that its methods are broadly applicable to other chemical transmitters that are expressed or released as a result of the activation of calcium signaling, including such transmitters that are identified in the future.
- the expression or release of such chemical transmitters can mediate the expression and/or release of other chemical transmitters.
- glutamate can induce the release of D-serine and ATP.
- ATP can induce the release of glutamate.
- ATP can induce the release of ATP.
- NO can induce the release of glutamate.
- PGE2 can induce the release of glutamate.
- the modulation of the expression or activity such proteins that participate in calcium signaling in an astrocyte can also be accomplished by means administering at least one antagonist to the subject.
- the modulation of the expression or activity of such proteins that participate in calcium signaling in an astrocyte can also be accomplished by means administering at least one agonist to the subject.
- Antagonists or agonists are preferably administered in therapeutically effective amounts, which will be known to those of skill in the art, or could be readily determined by routine empirical testing using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to _
- the effective amount of the agonist or antagonist may depend on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the neurological or psychiatric condition.
- a therapeutically effective dose of the antagonist or agonist will provide therapeutic benefit without causing substantial toxicity to the subject.
- the antagonist or agonist used is specific for a particular protein in the calcium signaling pathway, and non-specific antagonists or agonists that modulate the proteins can be used. Any agonist or antagonist for the calcium signaling pathway proteins in astrocytes that is now known in the art, or is later discovered is suitable for use in the methods of the present invention.
- Nonlimiting examples of antagonists useful in the methods of the present invention include: For mGluR5 receptors: 2-methyl-6-(phenylethynyl)- pyridine (MPEP); 3-[2-(methyl-l, 2-thiazol-4-yl)ethynyl pyridine (MTEP); [E]-2-Methyl-6-[2- phenylethenyljpyridine (SIB-1893); 6-Methyl-2-( ⁇ henylazo)-3-pyridinol (SIB-1757); for Gq: GpAntagonist-2A (GPant-2A); and, for phospholipase C: l-[6-[[17beta-3-methoxyestra- l,3,5(10)trien-17-yl]amino]hexyl]-lH- pyrrole-2,5- dione (U-73122), and 4-[[4-Formyl-5- hydroxy-6-methyl-3-[(phosphonooxy)methyl]
- the expression or secretion of the proteins involved in Ca +2 signaling can also be inhibited at the translation level.
- various agents can be used to target cellular protein expression machinery.
- the inhibitor may specifically suppress expression of calcium signal pathway proteins from the ribosomes, or may target expressed proteins for proteolysis, such as by ubiquitination of the proteins.
- Modulation of the expression of proteins that participate in calcium signaling in the astrocyte can also be by way of increasing expression of certain proteins in the pathway.
- increased expression of proteins can be carried out through overexpression of known regulatory proteins such as RGS2 and 1,4,5-trisphosphate (IP 3 ) phosphatase in the cell.
- RGS proteins are negative regulators of G protein-coupled signaling pathways, and the term "RGS" is deemed to encompass the over 20 RGS proteins that have been isolated to date, (see, De Vries L et al. (2000) Annu. Rev. Pharmacol. Toxicol. 40:235-71).
- IP 3 phosphatase negatively regulates IP 3 -mediated calcium release. Over expression of such proteins can be accomplished by means of genetic engineering, as set forth above.
- the genes encoding such proteins can be expressed under a strong promoter, a constitutive promoter, or an inducible promoter.
- the promoter can be tissue or cell specific, and can be specific for neurological tissues such as astrocytes or glial " cells. " Examples ' of strong promoters include the CMV promoter, SV40 early promoter, SV40 later promoter, SV2 promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, beta actin promoter, and other promoters shown effective for expression in eukaryotic cells.
- Nonlimiting examples of neurological tissue-specific promoters include the glial fibrillary acidic protein promoter (GFAP) (for astrocytes), and myelin basic protein promoter (for glial cells).
- Proteins can also be used to modulate the expression or activity of proteins that participate in calcium signaling in astrocytes.
- a polyclonal or monoclonal antibody that specifically binds to a protein that participates in calcium signaling in astrocytes such as a niGluR receptor, Gq, or phospholipase C can be used.
- any polypeptide that interacts with and/or modulates proteins that participate in calcium signaling in astrocytes can be used, e.g., a polypeptide that is identified using the presently described assays.
- any compound that is found to or designed to interact with and/or modulate the activity or expression proteins that participate in calcium signaling in astrocytes can be used e.g., a compound that is identified using the presently described assays.
- Any of the above-described molecules can be used to increase or decrease the expression or activity of proteins that participate in calcium signaling in astrocytes, or to otherwise affect the properties and/or behavior of proteins that participate in calcium signaling in astrocytes, e.g., stability, intracellular localization, interactions with other intracellular or extracellular moieties, and the like.
- inventive methods are applicable to treat or prevent any neurological or psychiatric condition that is induced, mediated, exacerbated, or sustained, in whole or in part, by calcium signal pathways in astrocytes, whether the condition is induced, mediated, exacerbated, or sustained by the astrocyte itself, or by the synapse, neuron, or other cell in which gliotransmission by an astrocyte produces an effect.
- Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP-17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
- ADHD attention deficit hyperactivity disorder
- Alzheimer's disease Parkinson's disease
- Huntington's disease depression
- hydrocephalus depression
- HIV dementia bipolar disorder
- FTDP-17 other tauopathies
- Hepatic encephalopathy Lupus encephalitis
- Also featured in accordance with the present invention are methods for treating or preventing a neurological or psychiatric condition in a subject which comprise modulating the expression or activity of one or more proteins that participate in the expression or release of glutamate.
- the methods are particularly applicable where the expression or release of glutamate initiates ' , ' mediates, facilitates, exacerbates, or sustains, in whole or in part, the neurological or psychiatric condition in the subject.
- the modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
- proteins that mediate glutamate release from astrocytes include, without limitation, synaptotagmin IV, synaptobrevin II, Cellubrevin, SNAP-23, Synataxin I, Munc-18, VGLUTl, VGLUT2, and VGLUT3.
- the modulation of the expression or activity of such proteins can be effectuated at the molecular level, including the nucleic acid or protein level, according to the methods described herein.
- the modulation of the expression or activity such proteins can also be accomplished by means of administering at least one antagonist or agonist to the subject.
- at least one antagonist or agonist to the subject.
- such antagonists or agonists are specific for at least protein that mediates glutamate release from astrocytes, but specificity is not critical, i.e., nonspecific antagonists or agonists can also be used.
- an agent administered to the subject inhibits the expression or activity of proteins that participate in glutamate expression or release by means of expression of the SNARE domain in order to prevent formation of the SNARE complex.
- the SNARE complex forms when four SNARE domains (soluble NSF attachment protein (SNAP) receptors), that are provided by three proteins come together to form a macromolecular protein complex.
- the proteins are comprised of a vesicular protein ⁇ e.g., synaptobrevin II) a plasma membrane protein ⁇ e.g., syntaxin) and a cytosolic protein ⁇ e.g., SNAP-25 in neurons, and SNAP-23 in astrocytes).
- Synaptobrevin and syntaxin each provide one SNARE domain, while SNAP-23 provides two.
- the expression of a dominant negative form of the calcium-sensing protein synaptogamin TV can also be used.
- This dominant negative form of synaptotagmin TV consists of mutations to the C2B domain, a putative Ca +2 -binding domain of Syt IV in which the first and the second aspartate residues of this domain were mutated to asparagines (D318,324N, referred to henceforth as B-D 1,2N), or the third aspartate and the fourth glutamate residues were mutated to asparagine and glutamine, respectively (D378N,E380Q, referred to henceforth as B- D3N,E4Q).
- Expression of these dominant negative molecules in astrocytes blocks the release of glutamate.
- Clostridial toxins especially the botulinum toxin and tetanus toxin, are highly specific proteases that cleave SNARE proteins.
- the quantity of each respective toxin that would need to be administered to the subject, and the frequency of administration can be empirically determined according to any method that is suitable in the art.
- Inhibition of the expression or activity of glutamate has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by glutamate expression or activity.
- Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP- 17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
- ADHD attention deficit hyperactivity disorder
- Alzheimer's disease Parkinson's disease
- Huntington's disease depression
- cerebrovascular disease hydrocephalus
- HIV dementia bipolar disorder
- FTDP- 17 other
- Also featured in the present invention are methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of one or more proteins that are activated by glutamate.
- the methods are applicable where the activation of the proteins by glutamate initiates, mediates, facilitates, exacerbates, or sustains, in whole or in part, the neurological or psychiatric condition in the subject. Activation can occur following the gliotransmission of glutamate.
- proteins that are activated by glial glutamate have been identified. Such proteins can be located in the synapse, proximal to the synapse, or even distal to the synapse. Such proteins outside of the synapse are generally referred to as extrasynaptic proteins.
- Non- limiting examples of proteins activated by glutamate include N-methyl-D-aspartate (NMDA) and ⁇ -amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) receptors. Glutamate can interact with any subunit on the NMDA receptor, including the NRI subunit, the NR2A subunit, or the NR2B subunit.
- Modulation of the expression or activity of proteins that are activated by glutamate can occur at the molecular level (nucleic acid or protein), as described herein. In some aspects, inhibition can be effectuated by means of administering at least one antagonist to the subject in a therapeutically effective amount.
- Nonlimiting examples of antagonists useful in the methods of the present invention include (i?)-2-ammo-5-phosphonopentanoate(D-AP5); (R)- AP5; (R)-CPP-ene; ( ⁇ )-c ⁇ -4-(4-phenylbenzoyl)piperazine-2,3-dicarboxylic acid (PBPD); (S)- ⁇ - amino-5-(phosphonomethyl)[l,r-biphenyl]-3-propanoic acid (EAB515); ( ⁇ )-6-(lH-Tetrazol-5- yl-methyl) decahydroisoquinoline-3-carboxylic acid (LY233536); Dizocilpine/(5S,10R)-(+)-5- Methyl-10,1 l-dihydro-5 ⁇ -dibenzo[a,d]cyclohepten-5,10-imine (MK-801); 3,5- Dimethyladamantan- 1 -amine (Memantine
- Modulation of the expression or activity of proteins activated by glutamate has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, exacerbated, or sustained, in whole or in part, by the activation of such proteins by glutamate in astrocytes or outside of astrocytes, such as in the synapse or extrasynaptic regions.
- the modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
- Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP- 17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
- ADHD attention deficit hyperactivity disorder
- Alzheimer's disease Parkinson's disease
- Huntington's disease depression
- hydrocephalus depression
- HIV dementia bipolar disorder
- FTDP- 17 other tauopathies
- Hepatic encephalopathy Lupus encephalitis
- the invention also features methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the oscillation of free cellular calcium.
- the methods are particularly applicable where the oscillation of the calcium mediates, in whole or in part, the neurological or psychiatric condition in the subject.
- the modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
- modulating oscillation of cellular calcium can occur via diminishing the availability of free calcium in the astrocyte.
- the availability of cellular calcium can be suppressed, for example, by use of a calcium chelator.
- Calcium cheltors are known in the art, and include, without limitation, EDTA, EGTA, diazo-2, FU ⁇ IA-2, and BAPTA-AM.
- Calcium chelators can be administered directly to the subject, for example, by injection into the cerebrospinal fluid of the ventricles of the brain, or onto the cerebral cortex. Calcium chelators can be injected in one bolus or could be continuously infused from a pump attached to a catheter, as is standard in medical practice.
- astrocytes are coupled in a syncitium by gap junctions, cells local to the injection site take up dye and then deliver it throughout the brain through this coupled network.
- This approach allows selective loading into astrocytes of BAPTA-AM, as well as other membrane permeant antagonists of chelating agents or AM coupled reagents.
- Modulation of the oscillation of free cellular calcium has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by the oscillation of free cellular calcium.
- Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HTV dementia, bipolar disorder, FTDP-17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
- ADHD attention deficit hyperactivity disorder
- ADHD attention deficit hyperactivity disorder
- Alzheimer's disease Parkinson's disease
- Huntington's disease depression
- cerebrovascular disease hydrocephalus
- HTV dementia
- D-serine is selectively synthesized and released from astrocytes. Without intending to be limited to any particular theory or mechanism of action, it is believed that decreasing the gliotransmission of D-serine will result in hypofunction of NMDA receptors. Accordingly, the invention feature methods to enhance D-serine release, and methods to enhance NMDA receptor function to treat or prevent neurological or psychiatric conditions. In some aspects, the methods are particularly applicable where the conditions are induced, mediated, exacerbated, or sustained, in whole or in part, by the diminished expression or activity of D- serine, or the diminished expression or activity of NMDA receptors.
- the modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated,- facilitated, exacerbated, or sustained by astrocytes.
- the methods comprise modulating the expression or release, i.e., gliotransmission, of D-serine, or modulating the expression or activity of the NMDA receptor
- the modulation of the expression or release of D-serine, or the modulation of the expression or activity of NMDA receptors can be effectuated at the molecular level, including the nucleic acid or protein level, according to the methods described and exemplified herein. It is preferred that the expression or release of D-serine, and/or the expression or activity of NMDA receptors is enhanced.
- the modulation of the gliotransmission of D-serine can also be facilitated by means of administering at least one agonist to a subject that has a neurological or psychiatric condition. Suitable agonists include, but are not limited to D-serine, cycloserine, and D-cycloserine.
- D-serine itself can be stimulated by various chemicals that induce Ca +2 oscillations in the astrocytes including glutamate, ATP, norepinephrine (NE), acetylcholine (Ach), and Bradykinin.
- Modulation of the expression or release of D-serine, and/or the expression or activity of the NMDA receptor has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by the gliotransmission of D-serine.
- Schizophrenia is a non-limiting example of such a condition.
- NMDA receptors can also be accomplished by means of administering at least one agonist to the subject.
- agonists are specific for the NMDA receptors, but specificity is not critical, i.e., nonspecific agonists can also be used.
- Non-limiting examples of NMDA receptor agonists include N- methyl-D-aspartate, l-aminocyclobutane-cis-l,3-dicarboxylic acid (ACBD), Homoquinolinic acid, and the like.
- the invention also features methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of one or more proteins that participate in the expression or release of ATP and/or adenosine. In these methods, adenosine expression or release ameliorates, in whole or in part, the neurological or psychiatric condition in the subject.
- a primary source of adenosine is the hydrolysis of extracellular ATP that is released ATP, which is hydrolyzed to adenosine (Pascual O et al. (2005) Science 310:113-6). It has been ,. Mu a K 11 discovered m accordance with the present invention that adenosine accumulates secondarily to the release of ATP from astrocytes.
- proteins have been identified as members of the pathways that facilitate adenosine expression or release.
- proteins include, but are not limited to bradykinin receptors, mGluR5, P2Y1, P2Y2, or noradrenergic receptors. Without intending to be bound to any particular theory or mechanism of action, it is believed that any receptors that are coupled to the action of phospholipase C induce the release of ATP.
- the methods comprise enhancing the expression or activity of these proteins. In another aspect, the methods comprise enhancing the expression or release of ATP.
- the methods can also comprise inhibiting the expression or activity of diacylglycerol kinase (DAGK) or diacylglycerol lipase.
- DAGK diacylglycerol kinase
- DAG lipase DAGL
- DAGK inhibitors include, but are not limited to, 6-[2-(4-[(4- fluorophenyl)phenyl-methylene]-l-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-alpha] pyrimidine-5-one (R59022), and 3-(2-(4-[bis-(4-fluorophenyl)methylene]-l-piperidinyl)ethyl)- 2,3-dihydro -2-thioxo-4(lH)quinazolinone (R59949), among others.
- Expression of the proteins that participate in the expression or release of ATP can be enhanced at the molecular level, using, for example, the methods described herein, or by using any of the various means for enhancing gene or protein expression that are known and routinely practiced in the art. Expression can also be enhanced by means of administering a therapeutically effective amount of an agonist of the expression of such proteins. For example, it has been discovered that administration of the DAG analog, l-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates ATP release from astrocytes.
- DAG analog l-oleoyl-2-acetyl-sn-glycerol
- OAG is one example of an agent that can be used with the inventive methods to facilitate ATP and thus, adenosine production.
- Additional agonists include, but are not limited to, DAG lipase inhibitor RHC-80267.
- the release of ATP itself may be stimulated by various chemicals that induce Ca +2 oscillations in the astrocytes including glutamate, ATP, NE, ACh, and Bradykinin.
- Facilitation of ATP and adenosine gliotransmission can also be achieved by inhibition of SNARE, as described and exemplified herein.
- Such methods are applicable to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by diminished levels or the absence of adenosine.
- modulation can be targeted to astrocytes, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
- the methods are applicable to treat or prevent any such condition that can be ameliorated by adenosine.
- adenosine has anticonvulsive properties (Lee KS et al. (1984) Brain Res. 321:160-4.)
- one preferred condition that can be treated or prevented using the methods of the invention is convulsions. Sleep abnormalities, anxiety, hypoxia and cerebral ischemia can also be ameliorated by enhancing adenosine release.
- Another aspect of the invention features methods to identify agents that modulate gliotransmission in astrocytes comprising contacting a test compound with an astrocyte and determining an increase or decrease in gliotransmission by the astrocyte in the presence of the test compound relative to the level of gliotransmission by the astrocyte in the absence of the test compound.
- the test compound can be assessed at multiple concentrations, and under varying environmental conditions such as temperature, oxygen, humidity, and the like.
- the assays can be carried out using freshly isolated astrocytes, or using astrocyte cell lines such as U373 MG, or C6 glioma.
- the effect of the test compound on gliotransmission can be carried out for any of the chemical transmitters produced by astrocytes, including without limitation, glutamate, adenosine, and D-serine, or any combination thereof.
- Determination of whether gliotransmission is increased or decreased in the presence of the test compound can be carried out using any means suitable in the art, which can vary depending on the needs of the investigator, or on which chemical transmitter(s) of interest is being evaluated.
- the assay used can be qualitative or quantitative. Those of skill in the art will know appropriate assays for determining gliotransmission of the various chemical transmitters, as well as how to fine tune such assays, all of which are routine in the art.
- the invention also features methods for identifying compounds that modulate gliotransmission by astrocytes using a combination of an in vitro and in vivo screening assay.
- a test compound is first screened in vitro as described herein, and then screened further in vivo to determine if the compound can modulate gliotransmission in the body.
- test samples are periodically taken from the subject and screened to determine if the test compound increased or decreased gliotransmission in neurological tissues within the subject. Once the test sample is taken from the subject, ex vivo screening on the test sample can be practiced according to the details described herein.
- the invention also features methods to identify agents that prolong neuronal cell survival.
- such methods are applied to identify agents that inhibit epileptogenesis in a subject.
- the methods comprise inducing status epilepticus in a test animal, . administering a test compound to trie test animal, and determining whether the test compound inhibits epileptogenesis in the animal relative to the frequency and severity of epileptogenesis in animals not treated with the test compound.
- Status epilepticus can be induced in a test animal according to any means that are suitable in the art.
- One preferred means is to administer pilocarpine to the test animal in an amount sufficient to evoke status epilepticus in the particular animal.
- the test animal can be any mammal, for example, mice, rats, rabbits, cats, dogs, non-human primates, guinea pigs, cows, horses, pigs, and the like.
- the test animal is a mouse.
- the amount of pilocarpine sufficient to evoke status epilepticus can vary depending on the type, sex, age, or weight of the animal, and may require empirical determination. Such empirical determinations are routine in the art.
- the seizure After induction of status epilepticus, the seizure is allowed to proceed for a period of time lasting for several minutes to several hours.
- the seizure can be allowed to proceed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, or more hours. Preferably, the seizure is allowed to proceed for 1 hour.
- the seizure is preferably terminated.
- the seizure can be terminated according to any means that are suitable in the art.
- the seizure can be terminated by administering to the animal diazepam in an amount suitable to terminate the seizure.
- the amount of diazepam sufficient to terminate the seizure can vary depending on the type, sex, age, or weight of the animal, and may require empirical determination, which are routine in the art.
- a test compound is administered to the animal.
- the test compound can be administered to the animal according to any means suitable in the art, such as by injection (intravenous, intramuscular, intradermally, and the like) orally, through inhalation, transdermally, enterically, and the like.
- the route of administration may vary according to the nature of the compound to be tested.
- the administration of the test compound can be intermittent or at a gradual, continuous, constant, or controlled rate.
- the time of day and the number of times per day that the test compound is administered can vary.
- the number of days in which the test compound is administered may vary.
- the test compound may be administered over a period of weeks, months, or years.
- test compound affects neuronal cell survival.
- screening methods of the invention will identify compounds that enhance as well as diminish neuronal cell survival, those compounds that prolong cell survival subsequent to status epilepticus are desired. Determination of whether the test compound prolongs cell survival can , be accomplished by screening neurological tissue for gliotransmission, particularly for glutamate, for calcium oscillation, or to determine whether glutamate binds the NR2B subuiiit of the NMDA receptor.
- Calcium oscillation can be measured according to any means suitable in the art.
- Non-limiting examples of such means include two photon calcium imaging and voltage clamping, as exemplified herein.
- Methods of measuring glutamate are known in the art and can include, for example, the use of glutamate biosensors or fluorometric methods (Neurosci Methods. 1991 Mar; 37(1):7-14. Neuroreport. 1997 May 27;8(8):2019-23). Methods for determining receptor binding are known in the art and are thus not described herein in detail.
- AU such measurements may be taken on any neuronal tissue, for example, isolated astrocytes or glial cells, or from brain slice preparations, as described and exemplified herein.
- this change in behavior of the Ca+2 signal has the potential to amplify synaptic inputs to a region of an astrocyte by releasing glutamate to hundreds of thousands of dendritic locales.
- the astrocyte amplifies local synaptic inputs through a cell-wide Ca+2 oscillation which stimulates glial glutamate to be released.
- Nimmerjahn et al (Nirnmerjahn et al., 2004) have demonstrated a simple method to load cortical astrocytes with small molecular weight Ca +2 indicators.
- small molecular weight compounds such as fluo4-AM or sulforhodamine 101 are topically applied to the cortical surface for 30-45 min.
- This loading technique results in the loading of astrocytes with these dyes in essentially 100% of animals and even in adult animals (>1 year of age).
- Surface loading selectively loads Ca +2 indicators into astrocytes, never into neurons ( Figure 2).
- the AM ester of the dye is injected into the cortex as described (see, Stosiek et al., 2003).
- Membrane-permeant dye e.g., fluo- 4AM
- the astrocytes typically exhibit relatively weak fluorescence intensity with this loading method. Without being limited to any particular theory or mechanism of action, it is believed that this low fluorescence is attributed to the diffusion of the dye from these locally loaded cells out into the astrocytic syncitium through gap junction connections.
- fluo- 4AM is injected into the cortex to label neurons, and then fluo-4AM and sulforhodamine are applied to the cortical surface to label the astrocytes.
- Neurons are loaded with Ca +2 indicator only while astrocytes are co-loaded with fluo-4 and sulforhodamine 101 ( Figure 3).
- a two-photon microscope can be used to study astrocyte-neuron interactions.
- This two-photon microscope integrates two scan control mechanisms, one to control the imaging and the other the photolysis pathway.
- the two-photon microscope is coupled to two Ti:Sappliire lasers, one of which is tuned to 720nm for photolysis, and the other of which is tuned to 820nm to excite fiuo-4 for imaging.
- Figure 4 shows the changes in Ca +2 level in an astrocyte and demonstrates that photo-release of nitric oxide (NO) from caged NO induces a Ca +2 elevation, which results from the influx of Ca +2 (Li et al. , 2003).
- This capability can be used to integrate two-photon photolysis with imaging to study the ability of the astrocyte to release glutamate and activate NR2B-containing NMDA receptors.
- FVB/NJ mice were anesthetized with urethane (1.5-2.0 mg/g body weight) and held in an immobilization device. Next, a circular cranial window (2.0 mm diameter) was drilled in the skull overlying the cortex. A metal frame, to attach the skull to the microscope platform, was attached to the skull using cyanoacrylate, and the dura was carefully removed.
- the fluorescent signals were quantified by measuring the mean pixel intensities of the region of interest using Metamorph software (Universal Imaging Corp). Ca +2 changes were expressed as ⁇ F/F 0 values, where F 0 was the baseline fluorescence. To express the magnitude of Ca+2 signals without subjective selection of threshold values, the ⁇ F/F 0 signal was integrated over a 300s imaging period using Origin software.
- Whisker-evoked potentials were recorded using a glass electrode filled with ACSF and connected to a Swam HC amplifier (Celica, Slovenia). Mouse whiskers were stimulated with a 100ms air puff through a glass tube while EP was being recorded. Data were acquired with a Digidata 1320 interface and pClamp software (Axon Instruments, CA). Brain Slice Preparation and Patch Clamp Aecordings and Analysis
- Transverse brain slices 300-400 ⁇ m were prepared from FVB/NJ mice (Jackson lab, Bar Harbor, Maine) at postnatal days 12-45 as described previously (Pasti et ah, 1997). After cutting, slices were incubated at 37° C for a recovery period of lhr. The solution for slice cutting and incubation was (in mM): NaCl 120, KCl 3.2, NaH 2 PO 4 1, NaHCO 3 26, MgCl 2 2, CaCl 2 1, glucose 2.8, Na-pyruvate 2 and ascorbic acid 0.6 at pH 7.4 with O 2 95 %, CO 2 5%.
- Patch clamp recordings were performed using standard procedures using MultiClamp-700B amplifiers (Axon instruments, Union city, CA) or 2400 patch-clamp amplifiers (A-M Systems Inc., Sequim, WA). Data were filtered at 1 KHz and sampled at 5 KHz with a Digidata 1320 interface and pClamp software (Axon Instruments). Experiments were performed at 30-35°C. Neurons were voltage-clamped at -60 mV. Evoked postsynaptic currents (EPSCs) were elicited by intracortical stimulation (0.1 Hz) with a bipolar tungsten electrode placed 100-150 ⁇ m from the recording pipette.
- Evoked postsynaptic currents Evoked postsynaptic currents (EPSCs) were elicited by intracortical stimulation (0.1 Hz) with a bipolar tungsten electrode placed 100-150 ⁇ m from the recording pipette.
- the rise time of the NMDA component of the EPSCs and SICs was calculated with the 20-80% criterion and the decay time as the time constant of a single exponential fit. Data analysis and fitting were performed with Clampfit 9.2 (Axon Instruments), Origin (Microcal Software, Northampton, MA) and SigmaPlot 8.0 (SPSS Inc., Chicago, IL) software.
- methyl scopolamine (a muscarinic antagonist) was administered subcutaneously (s.c.) (1 mg/kg) to reduce adverse, peripheral affects.
- Status epilepticus was induced through s.c. injection of 350 mg/kg piloca ⁇ ine-hydrochloride, a muscarinic agonist. Animal behavior and seizure activity was documented throughout the procedure.
- seizure activity was reduced by the administration of diazepam (5mg/kg) subcutaneously.
- Control mice two groups were injected either with saline or with a l/10 th dose of pilocarpine.
- MPEP and ifenprodil were administered intraperitoneally (i.p.) to mice together with diazepam.
- Antagonists were subsequently administered i.p. once daily for up to 3 days before euthanization.
- Fluoro-Jade B staining brain sections were washed with PBS 3 times and then immersed into 0.0001% Fluoro-Jade B in 0.1% acetic acid solution at 4 0 C for 1 hr.
- double staining sections were stained with either ApopTag kit (UpState) or biotinylated mouse anti-NeuN (1 : 100) followed by Fluoro-Jade B staining procedure as described above. All sections were counterstained with Dapi (1 : 1000, in PBS) and mounted using anti-fade mounting medium. Stained sections were viewed with epifluorescence and double-stained sections were examined using an Olympus fluoview 1000 confocal microscope and analyzed using Metamorph (Universal Imaging Corp).
- FJB-labeled cells were counted bilaterally by a blind investigator. To count the number of neurons in area C A3 and cortex cells images of DAPI and NeuN staining in area CA3 of the hippocampus and 70-150 ⁇ m beneath the surface of cortex were acquired with a digital camera. Automated software was used to identify and count neuronal cell bodies based on the co- localization of NeuN with DAPI. Manual inspection confirmed the accuracy of this automated approach., and analyzed.
- N values reported in in vivo studies represent the number of animals.
- Ca +2 measurements were generally made from 6- 10 astrocytes per animal, and the data were averaged to obtain a single value per animal.
- neuron and FJB counting studies measurements were made on at least four sections which were used to obtain a single value representative of that animal.
- ANOVA tests were performed and differences between individual groups were determined using the Newman-Keuls post- hoc tests. Electrophysiological studies were tested for significance using the student's t-test unless stated otherwise. Statistical significance of data was reached at p ⁇ 0.05.
- astrocytes in the barrel cortex were selectively loaded with the Ca +2 indicator Fluo-4 two or more days after pilocarpine-induced status epilepticus (Fig 5A).
- Ca +2 oscillations were synchronous between adjacent astrocytes (Fig. 5G- J) with large correlationcoefficients at zero lag time (Fig. 5H, I).
- niGluRj mediates enhanced astrocytic calcium oscillations following status epilepticus. Because niGluR5 contributes to astrocytic Ca +2 oscillations in brain slices, whether this receptor mediates the enhanced astrocytic Ca +2 signaling detected following status epilepticus was investigated. First, it was determined whether mGluR5 agonists would mimic in control mice the enhanced Ca +2 excitability detected following status epilepticus.
- DHPG 3,5-Dihydroxyphenylglycine
- CHPG 2-chloro-5- hydroxyphenylglycine
- a selective mGluR5 agonist induced enhanced Ca +2 signaling within astrocytes
- CHPG-induced Ca +2 signals are due to the selective activation of mGluR5 because they are prevented by the mGluR5 antagonist MPEP (30 ⁇ M; Fig. 7B,C).
- mGluR5 -dependent activation of astrocytic Ca+2 signaling evokes NR2B- dependent NMDA receptor-mediated neuronal currents.
- astrocytic Ca +2 oscillations to cause glutamate-mediated neuronal excitation, as has been described for thalamic and hippocampal pyramidal neurons (Angulo et at, 2004; Fellin et ah, 2004; Perea and Araque, 2005)
- studies in acutely isolated brain slices were performed to determine whether layer 2/3 cortical astrocytes excite cortical pyramidal neurons.
- the class I mGluR agonist DHPG (10-20 ⁇ M) as well as the mGluR.5 selective agonist CHPG (0.5-1 mM) induced TTX insensitive SICs in layer 2/3 cortical neurons that were blocked by the mGluR5 antagonist MPEP (50 ⁇ M; Fig. 9 A, B; n 17, p ⁇ 0.05).
- SICs fulfill the remaining pharmacological criteria to assign their origin to the astrocyte: they are mediated by the selective activation of NMDARs since they are reversibly blocked by DAP5 (50 ⁇ M, Figure 9C) while NBQX (10 ⁇ M) does not change the amplitude and kinetics of SICs (Tablel).
- SICs are significantly slower than NMDA receptor mediated EPSCx and SICs are independent of AMPA receptor activation. Mean rise, decay time, and amplitude for SICs and intra-cortical- evoked NMDAR EPSCs.
- intra-cortical NMDA receptor-mediated EPSCs were reversibly attenuated by NVP-AAM077 (Fig. 9G-H; p ⁇ 0.01) but insensitive to Ifenprodil (Fig. 9F,H).
- the selective attenuation of synaptic NMDA receptors by NVP-AMM077 and attenuation of SICs by ifenprodil show that gliotransmission, that is mediated by SICs, selectively accesses extrasynaptic NR2B-containing NMDA receptors.
- MPEP and ifenprodil can be used selectively to prevent astrocytic Ca +2 oscillations and to attenuate astrocyte-evoked SICs without significant actions on either synaptic NMDAR currents or on whisker-evoked synaptic potentials and neuron-based integration.
- NVPAAM077 which attenuates NMDAR-mediated synaptic transmission, did not provide neuronal protection (Fig. 1OD, E).
- MK801 a use-dependent NMDAR antagonist which does not select between NR2B- or NR2A-containing NMDA receptor subunits offered minor protection.
- FJB labeling seven days were allowed to elapse following status epilepticus, after which NeuN-positive neuronal cell bodies were identified and counted.
- astrocyte- evoked neuronal NMDA currents may be a generalized phenomenon of the nervous system as these events have been detected in each region of the nervous system that has been tested thus far, including areas CAl and C A3 of the hippocampus, cortex and nucleus accunibens.
- NR2B-containing receptors are known to be at extrasynaptic locales after synapses have developed (Tovar and Westbrook, 1999; Rumbaugh and Vicini, 1999) this important observation provides further support for the notion that glial glutamate acts on extrasynaptic NMDA receptors. Additionally, activation of NR2B-containing NMDA receptors can activate a neuronal cell death pathway.
- mice were administered either MPEP (20mg/kg) or ifenprodil (20mg/kg) i.p. on each of three days. Animals were then cardiac perfused, and brains were sectioned and stained with Fluoro-Jade B which labels dying neurons (Schmued and Hopkins, 2000). In parallel controls, Ca +2 oscillations in cortical astrocytes were imaged, confirming that they were at control levels following i.p. MPEP administration.
- RGS proteins regulate GTP binding signaling by terminating the active G protein signal (Hains et al, 2004). Receptor occupancy by an agonist causes the exchange of GDP for GTP and dissociation of G ⁇ . CT p from the ⁇ / ⁇ subunits. G ⁇ . G ⁇ p and ⁇ act on effectors to stimulate signaling. Termination of the signal is regulated by the GTPase activity of Ga which is controlled by RGS proteins. Overexpression of RGS proteins leads to an abrupt termination of the receptor activated signal.
- RGS2 interacts with Gq ⁇ the ⁇ -subunit that is predominantly responsible for mediating the activation of phospholipase C, and thus the generation OfIP 3 and Ca +2 release from internal stores, by a variety of receptors including niGluR5 and purinergic receptors such as P2Y 1 and P2Y 2 .
- RGS2 The expression in astrocytes of RGS2 can perturb receptor-induced PLC and IP 3 -dependent Ca +2 signaling.
- RGS2 can be expressed together with DSRed2 in astrocytes and assayed ATP-induced Ca +2 signaling.
- RGS2 potently blocks G q signaling pathways as ATP, even at concentrations as high as lOO ⁇ M (receptor K D ⁇ l ⁇ M), was unable to evoke Ca +2 signals compared to DsRed2 expressing cells.
- RGS2 has been investigationally introduced into adenoviral and lentiviral vectors that have been engineered to contain the GFAP promoter for astrocyte-specific expression of RGS2.
- this viral approach can be used to introduce RGS2 into astrocytes to determine whether this cell type-specific uncoupling of gliotransmission will reduce delayed excitotoxicity following status epilepticus.
- the cytosolic portion of the SNARE domain of synaptobrevin 2 was selectively expressed in astrocytes, a manipulation that blocks gliotransmission.
- Two lines of transgenic mice were developed. In the first, GFAP.tTA, the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter drives the expression of the Btet-Off tetracycline transactivator (tTA).
- the second, tetO.SNARE contains a tet operator (tet ⁇ )-regulated SNARE domain and lacZ and enhanced green fluorescent protein (EGFP) reporter genes (Fig. 13A).
- Crossing lines yields mice in which SNARE, LacZ, and EGFP transgenes are expressed in GFAP-positive astrocytes, not in neurons (Fig. 13B and E to J), and in which transgene expression is suppressed by doxycycline (Dox) (Fig. 13, C and D). These animals are referred to as dominant negative SNARE (dn-SNARE) mice.
- dn-SNARE dominant negative SNARE
- astrocyte-specific expression of the dn-SNARE domain affects synaptic transmission and plasticity by using acutely isolated hippocampal slices.
- fEPSPs Schaffer collateral-evoked field excitatory postsynaptic potentials
- LTP long-term potentiation
- D-AP5 D-2-amino-5 phosphonopentanoate
- NMDA N-methyl-D-aspartate
- glial glutamate preferentially activates NR2B subunit-containing NMDA receptors
- an NR2B subunit-containing NMDA receptor antagonist was tested and revealed actions on neither the fEPSP slope (Fig. 14E) nor LTP (Fig. 14G).
- PPADS pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid
- RB-2 reactive blue-2
- ATP can be converted into adenosine by ectonucleotidases. Because there is a tonic level of extracellular adenosine that acts through the Al receptor to persistently suppress excitatory synaptic transmission, whether adenosine mediated the effects of astrocyte- specific dn-SNARE expression was evaluated.
- the Al receptor antagonist 8-cyclopentyl-l,3- dipropylxanthine (DPCPX) 800 nM
- Tetanic stimulation of Schaffer collaterals causes an adenosine-mediated, heterosynaptic depression of neighboring unstimulated synapses.
- the source of the adenosine remains an enigma.
- stimulation of the Schaffer collaterals induces Ca signals in astrocytes, which in turn evoke the release of gliotransmitters, and because heterosynaptic suppression has been indirectly linked to a glial-dependent mechanism
- the dn- SNARE mice were used to determine whether astrocytes mediate heterosynaptic depression. Two independent pathways (Sl and S2) were each stimulated at 30-s intervals to monitor baseline synaptic transmission (Fig. 16A).
- this study was repeated using dn-SNARE slices and found a virtual absence of heterosynaptic depression (Fig. 16C).
- the kinetics of ATP hydrolysis and adenosine accumulation provide a synaptic network with unique spatiotemporal conditions to control synaptic transmission.
- Fast-acting synaptic transmitters such as g-aminobutyric acid and glutamate have high-affinity uptake systems in the vicinity of the synapse that constrain the time and distance over which a transmitter acts.
- Synaptic activation of an astrocyte to release ATP removes these constraints, because it takes -200 ms before adenosine begins to accumulate. This provides time for ATP diffusion to distant sites, where it depresses synaptic transmission through accumulated adenosine, thereby providing a mechanism for cross-talk to distant synapses.
- astrocytes In addition to activity-dependent actions, astrocytes, by persistently suppressing excitatory synaptic transmission, enhance the capability of synapses to express synaptic plasticity. Thus, the integration of synaptic activity by the astrocyte leads to a widespread coordination of synaptic networks. By suppressing excitatory transmission, astrocytes regulate the degree to which a synapse maybe plastic, and during the induction of LTP, astrocyte-derived adenosine depresses neighboring unstimulated pathways.
- astrocytes may need to be relocated to determine whether the addition of agonists and antagonists affect their Ca +2 signaling.
- rhodamine dextran can be injected into the vasculature to identify specific volumes in , doctor ⁇ • i i the brain, much like one uses a road.
- Figure 17 shows that with this approach, the same individual astrocytes can be identified and relocated such that astrocytes in different brain volumes can be imaged before a pharmacological agent is provided, and then afterwards to dete ⁇ nine the resultant changes in signaling.
- 24h monitoring can be performed to select mice that have not had a seizure during the previous day (to prevent acute effects of a seizure) for the imaging of astrocytic Ca +2 . With these animals it can be determined whether astrocytic Ca +2 signaling, and thus gliotransmission, are enhanced in the hippocampus of epileptic mice.
- Ca +2 signals can be measured within processes as well as the cell body to determine the degree to which Ca +2 signals are coordinately regulated within a cell by performing a cross correlation analysis of the Ca +2 signals within distinct process of the cell before and after addition of DHPG.
- the goal of adding DHPG is not to fully activate mGluR5, but instead to provide a partial activation of these receptors on which subsequent inputs can summate. Therefore, a dose response relationship should be performed to determine the minimal concentration of DHPG required to switch local to global Ca +2 signals.
- the mGluR5 selective antagonist MPEP can also be applied, which will determine whether the observed effects result from mGluR5 activation.
- an alternative strategy is to provide focal stimulation, through a small extracellular pipette, to a small group of Schaffer collateral afferents adjacent to the processes of an astrocyte.
- focal stimulation through a small extracellular pipette, to a small group of Schaffer collateral afferents adjacent to the processes of an astrocyte.
- local Ca +2 signals can be evoked wherein it can be investigated whether increasing concentrations of DHPG increase the range over which these astrocytic Ca +2 signals spread.
- photolysis is used to stimulate a local region of cellular processes and determine the extent to which the Ca +2 signal spreads through the arbor of the astrocyte. Given the evidence of the capacity to photo-release caged compounds in vivo, the goal is to perform this experiment entirely in vivo.
- Caged MNI-glutamate can be introduced into the cortex in vivo, either through application to the cortical surface or through a focal ejection from a pipette, and can focally photo-release glutamate onto a region of the astrocytic processes to evoke a local Ca +2 signal.
- the extent of spread of the Ca +2 signal can then be determined to confirm that it is due to activation of mGluR5 by co-application of MPEP (tail vein injection).
- glutamate can be photoreleased and, after determining the extent of spread of the Ca +2 signal, the cortical surface DHPG can be applied at increasing concentrations from 1 to 50 ⁇ M, although the exact concentration may vary depending on the results of the experiments detailed in Example 7.
- DHPG increases the range of spread of the photolysis-evoked Ca +2 response. Additionally, a glutamate dose-response curve should be performed in which the magnitude of the local Ca +2 signal in relation to the photolysis power is determined, and to determine whether concentrations of DHPG that alter the spread of the Ca + signal also reduce the power required to evoke a Ca +2 signal.
- concentrations of DHPG that alter the spread of the Ca + signal also reduce the power required to evoke a Ca +2 signal.
- Such studies should be performed at constant depth in the cortex to set forth a constant amount of scattering of excitation energy between different experiments. It is anticipated that low concentrations of DHPG will enlarge the distance over which focally-released glutamate will evoke a Ca +2 signal.
- a disadvantage of the approach may be the experiments performed in brain slices to allow reliable introduction of caged compound by a patch pipette into an astrocyte.
- caged IP 3 and caged MNI-glutamate caged serotonin can be used. With caged serotonin, studies will be able to be performed in vivo having independent control over the receptor activated by photolysis and by DHPG application.
- caged IP 3 (delivered from a patch pipette) will be focally released into the interior of an astrocyte. It can then be determined whether subsequent addition of MPEP reduces the extent to which photolysis evokes a Ca +2 signal. If there is an elevated basal extracellular glutamate level, then it is predicted that this will lead to an overall increase in resting IP 3 level in the astrocyte. Though this small change may be sub-threshold for inducing significant Ca +2 signals, when it is additive with a pulse of IP 3 it is - 11 IL... i / a / ⁇ '..:;: . ( ;; predicted that Ca may be released over a larger region of the cell. As an alternative to caged
- IP 3 caged serotonin is available, which can be used in vivo.
- neuronal death is detected in layer 2/3 cortical neurons and in area C A3 of the hippocampus, it is essential to determine whether astrocyte-evoked SICs are present in these locations in addition to area CAl and to further determine whether the NR2B-containing NMDA receptor mediates these currents.
- Brain slices were prepared from control animals and paired whole-cell recordings were made from pyramidal neurons to monitor for astrocyte-evoked SICs while simultaneously imaging astrocytic Ca +2 . While recording from neurons the characteristic time- course of SICs, which exhibit a slower rise time than synaptic currents ( ⁇ 70ms compared to ⁇ 2ms) as well as a slower decay time (450ms compared to ⁇ 10ms) were monitored. Under normal physiological conditions, astrocyte-evoked SICs were detected only in about 30% of the neurons and at a low frequency of about one event every 10 min.
- the mGluR was superperfused agonist DHPG (lOuM), which in area CAl increases cell-wide astrocytic Ca +2 oscillation frequency, to determine whether cell-wide astrocytic Ca +2 oscillation frequency and SIC frequency were augmented. Additionally, it was determined whether the actions of DHPG are mediated by mGluR5 by co-applying, in some experiments, the mGluR5 antagonist MPEP (5OuM).
- SICs were blocked by the NMDA receptor antagonist D-AP5, augmented in frequency and magnitude by removal of external Mg +2 , and sensitive to the NR2B subunit containing NMDA receptor antagonist ifenprodil (Fellin et al, 2004).
- Table 2 shows the distinctive pharmacological characteristics that, together with their distinctive kinetics, allow the detection of SICs as arising from an astrocyte source. These pharmacological tools confirmed the NR2B subunit containing nature of the NMDA response of SICs.
- DHPG permits cell-wide astrocytic Ca +2 oscillations in layer 2/3 of the cortex in vivo, it is anticipated that low concentrations of DHPG will allow microdomam Ca +2 elevations to become cell-wide events and will thus increase the frequency of observing large amplitude astrocyte-evoked neuronal SICs.
- a 3 ⁇ m spot of UV energy can be illuminated onto a cell body of an adjacent astrocyte, and the relative Ca +2 level can be monitored using fluo-4 thereby permitting the determination of when the Ca +2 signal propagates to the region of the dendrite.
- Simultaneous electrophysiology can be used to identify the presence of astrocyte-evoked currents. Since previous studies have shown that SICs can be repetitively evoked, prior to subsequent photolysis pulse, antagonists as ifenprodil and D-AP5 can be included in the superfusate to determine whether CA3 and cortical layer 2/3 astrocytes evoke NR2B subunit-containing NMDA responses in adjacent pyramidal neurons.
- Brain slices obtained from transgenic animals, wild-type littermates, or from wild-type animals previously injected with adenovirus to induce the expression of RGS2-EGFP under the control of a GFAP promoter can be used to simultaneously study the ability of the mGluR5 agonist to induce cell-wide astrocytic Ca +2 signals as well as neuronally detected NMDA receptor-dependent SICs. Because preliminary studies have already shown significantly reduced glutamate release in cultures from the astrocyte-specific SNARE animal, it is anticipated that the resting and DHPG-induced frequency and amplitude of SICs will both be reduced when compared to wild-type littermates and transgenics maintained on doxycycline to suppress the expression of the dominant negative SNARE domain. In contrast, it is expected that the expression of the transgene will have no impact on the astrocytic Ca +2 signal as this is upstream of the glutamate release machinery.
- RGS2-EGFP When RGS2-EGFP is virally expressed in astrocytes, it is expected to block, or attenuate, the ability of DHPG to evoke a Ca +2 response in astrocytes, and will similarly prevent astrocyte-dependent Ca +2 -evoked neuronally detected SICs.
- the Ca +2 signal and SIC parameters can be compared to those recorded in adjacent slices from the same animal that were not infected by the virus and additionally compared to control animals that received viral injections in which GFAP promoter regulates EGFP expression only.
- the Al receptor antagonist DPCPX (80OnM) can be included in experiments using wild-type and transgene-expressing slices so that, regardless of whether the transgene is expressed, the persistent Al activation is absent.
- the Al agonist, CCPA (10-5OnM) can be included in the ACSF to recover the persistent Al activation in transgene-expressing slices.
- a second potential concern is that transgene expression is weak in the cortex relative to the hippocampus. Therefore, in experiments focused on the cortex it is important to determine whether sufficient transgene is expressed in astrocytes to block glutamate release from astrocytes. If no effect on glial glutamate release is observed, the viral expression of RGS2 alone can be used in cortical experiments. Indeed, because multiple strategies to manipulate glial glutamate release, there is some redundancy built into the experimental design to overcome potential problems. If RGS2 is determined to be expressed at levels insufficient to inhibit G q - mediated, astrocytic Ca signaling, a backup with mutant Gq ⁇ subunits is available. In one of these, the Gq ⁇ is in a persistently GTP-bound form. Because of the persistent activation, it is believed that internal Ca +2 stores are depleted leading to a blockade of receptor-dependent astrocytic Ca +2 signaling.
- a third potential concern is how widespread transgene expression will be following adenovirus injection and whether there might be cytotoxic effects from the adenovirus.
- success has been obtained with the adenovirus technique, astrocyte-specific lentiviral vectors are currently in development that can serve as alternatives.
- Tetracycline- regulated lentivirus constructs that permit the use of doxycycline to control the expression of the genetically encoded Ca +2 indicator, VC6.1, have already been developed to study transplantation of stem cells into the cortex.
- an alternative strategy is to develop a tetracycline-regulated RGS lentiviral construct.
- the existing tetO promoter can be used, and, after introducing RGS-EGFP, the virus can be injected into the hippocampus and cortex of transgenic animals in which the GFAP promoter drives the expression of the transactivator, tTA, selectively in astrocytes.
- This can provide cell-type specific, doxycycline regulation of the transgene RGS2. In this manner there can be extraordinar control over transgene expression by addition of doxycycline to the animals' drinking water, which will allow the control of potential cytotoxic effects of viral injection.
- membrane receptors can be internalized during status epilepticus, it is important to determine whether each of the signaling elements in the gliotransmission pathway is intact following status epilepticus. Since it has been determined that the astrocytic Ca +2 signaling pathway is functional, whether astrocytic Ca +2 signals stimulate glutamate release detected by neuronal NR2B-containing NMDA receptors was evaluated.
- Brain slices were isolated from animals 1-3 days following status epilepticus and perform experiment similar to those Examples above. Spontaneous and DHPG-evoked astrocytic Ca +2 signals and the presence of SICs were recorded. As a follow-up, whether SICs are D-AP5 and ifenprodil sensitive was detennined, along with whether the expression of the dominant negative SNARE domain in astrocyte blocks this pathway, and whether the expression of RGS2 blocks astrocytic Ca +2 signaling and SICs. By performing this experiment on tissue isolated from animals 1-3 days following status epilepticus, the e ⁇ ectiveness of the reagents that used to perturb this pathway in vivo was determined in addition to demonstrating the presence of gliotransmission. ⁇
- SICs can be identified on the basis of their kinetics. Sensitivity to D-AP5 and ifenprodil and insensitivity to TTX and CNQX can be used to identify SICS. Measurements are made in ACSF (rest) during application of DHPG, and during application of the niGluR5 antagonist MPEP. It is predicted that there will be an elevated frequency of Ca +2 oscillations under resting conditions due to cell-wide Ca +2 oscillations, and that DHPG will have little further stimulatory effect on either astrocytic Ca +2 or SIC frequency. It is further predicted that MPEP will reduce the frequency of both the Ca +2 oscillation and the SIC.
- the seizure is terminated by injection of diazepam.
- the test pharmacological agent can be injected into the animal, and the animal returned to its cage.
- the antagonist can be re-injected into the animal at successive daily intervals, for a total of four injections. It has previously been determined that this injection protocol significantly reduces neuronal death. Animals are to be sacrificed at 1, 3, 5, 7 and 10 days following status epilepticus (5 animals in each group). The animals can then be cardiac- perfused with 4% paraformaldehyde, and sections of cortex and hippocampus (20um) can then be cut and reacted with Fluoro-Jade B (Schmued and Hopkins, 2000) to disclose dying cells.
- ifenprodil prevents the increase in astrocytic Ca +2 oscillations that normally follow status epilepticus. Since ifenprodil acts downstream of gliotransmission on the target of glial glutamate action, the NR2B-containing NMDA receptor, acute administration of ifenprodil does not reduce astrocytic Ca +2 oscillations when administered at the time of imaging astrocytic Ca +2 three days following status epilepticus. However, it should be determined whether daily treatment with ifenprodil, which preliminary experiments suggest significantly suppresses delayed neuronal death, prevents the increase in astrocytic Ca +2 oscillations that are normally seen following status epilepticus.
- Example 25 It is expected that the experiments in Example 25 will provide significant new insights into the role of mGluRs and NR2B-containing NMDA receptors in mediating delayed neuronal death. While these studies should implicate the astrocyte as a contributor to neuronal death, unequivocal identification of such a role will require astrocyte-specific manipulations to prevent the activation of gliotransmission. Thus, astrocyte-specific transgenic animals and virally expressed RGS2 can be used to block astrocyte-glutamate release and G q -evoked Ca +2 signaling respectively, in order to confirm that astrocytes contribute to delayed neuronal death.
- transgenes will be expressed in astrocytes to block glutamate release and G q -dependent Ca +2 signaling, instead of injecting antagonists after status epilepticus and determining consequences for delayed neuronal death. Because it may not possible to instantaneously turn-on or off the expression of the transgene. Thus it may be necessary to ;;ii. , ⁇ i . , . , . ., . control the expression of the transgene prior to the induction of status epilepticus then determine consequences of astrocyte-specific transgene expression on delayed neuronal death. The exact conditions for the best expression of these transgenes is discussed in Example 22, above.
- astrocytic Ca +2 signals can be measured following status epilepticus to determine whether RGS2 expression suppresses cell-wide Ca +2 oscillations and to determine whether the inhibition of gliotransmission by SNARE domain expression in astrocytes prevents the augmentation of Ca +2 signaling. If SNARE domain expression blocks or reduces the increase in Ca +2 signaling together with reducing delayed neuronal death, this would be consistent with a positive feedback control of glial Ca +2 signaling as discussed in Example 25 when ifenprodil is administered to animals.
- astrocyte-specific SNARE domain expression retards neuronal death and reduces the increase in Ca +2 signaling normally detected in animals following status epilepticus this would suggest that an initial neuronal damage event increases astrocytic Ca +2 signals which in turn feed back to stimulate further neuronal damage.
- Anti-GFAP immunoreactivity can be used to confirm that transgenes are selectively expressed in astrocytes.
- DPCPX (lmg/kg) or the Al agonist CCPA (lmg/kg)
- CCPA the concentration of pilocarpine
- osmotic mini-pumps can be used for administration of pharmacological agents into the nervous system. Pumps can be used for sustained administration of Al pharmaceuticals, as well as for glutamatergic pharmaceuticals, as appropriate, since this approach should prevent unwanted systemic effects of pharmaceuticals.
- Pilocarpine is injected into mice to evoke status epilepticus, and after one hour, the seizure is terminated by injection of diazepam. After a further one hour period the test pharmacological agent (e.g., MPEP and ifenprodil) is injected into the animal, and the animal is returned to its cage. At daily intervals, the antagonist can be re-injected for between three and seven days. Animals can be maintained for 30-90 days, and the degree to which the animals develop spontaneous seizures can be assessed.
- the test pharmacological agent e.g., MPEP and ifenprodil
- mice that selectively express a dnSNARE transgene under control of a tetracycline regulator only in astrocytes were used to study the release of astrocyte-dependent accumulation of adenosine in epileptic animals.
- Animals were maintained on the tetracycline analog Doxycycline to prevent dnSNARE expression (Figure 18). Animals were maintained in this manner, then status epilepticus was induced and animals maintained until at least one month later when they became epileptic. At this point animals were videotaped to monitor seizure frequency. Data are shown for three sequential analysis periods, performed within the same animals.
- seizure frequency was monitored while on a diet containing doxycycline to prevent transgene expression. Doxycycline was then removed, to allow dnSNARE expression, seizure frequency again assessed, then doxycycline was added back to the diet to prevent dnSNARE expression and again seizure frequency was assessed.
- the seizure frequency was found to increase when astrocytes did not regulate adenosine accumulation (doxycycline removed from diet and dnSNARE expressed in astrocytes). This result clearly demonstrates that the regulation of accumulation of the natural anticonvulsant adenosine from astrocytes controls epileptic seizures. It should be noted that in this experiment treatments began after epilepsy had commenced and that selective manipulation of astrocytes and their control of adenosine accumulation can be used to control seizures.
- FIG. 19 shows that treatment of the cortex with BAPTA/AM, which selectively chelates Ca +2 in astrocytes, attenuates neuronal death as assayed by FJB.
- BAPTA/AM selectively loads into astrocytes to chelate their internal Ca +2 and prevent Ca +2 oscillations and gliotransmission this data provides a direct demonstration that astrocytes are responsible for the delayed neuronal death that follows status epilepticus.
- Microdomains for neuron-glia interaction parallel fiber signaling to Bergmann glial cells. Nat
- mGluR3 and mGluR5 are the predominant metabotropic glutamate receptor mRNAs expressed in hippocampal astrocytes acutely isolated from young rats. J Neurosci Res 58: 533-543.
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Abstract
The present invention features novel methods for treating neurological and psychiatric conditions. The inventive methods comprise modulating the production or activity of one or more proteins that participate in calcium signaling or glutamate release in astrocytes, modulating the production or activity of one or more proteins that regulate the action of glial glutamate, modulating the concentration of calcium in the neuronal cell, modulating the expression or release of D-serine, or modulating the expression or release of ATP or adenosine. Methods to screen test compounds for their ability to target the specified pathways or cellular calcium are also disclosed.
Description
METHODS FOR TREATING NEUROLOGICAL AND PSYCHIATRIC CONDITIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit to United States Provisional Application No. 60/692,513, filed June 21, 2005, the entire contents of each of which are incorporated by reference herein, in their entirety.
GOVERNMENT SUPPORT
[0002] Research leading to the disclosed inventions was funded, in part, with funds from the National Institutes of Health, grant nos. R01-NS-054770, R37-NS-037585, ROl-NS- 043142, and P20-MH-071705. Accordingly, the United States government may have certain rights in the inventions described herein.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of neuropharmacology. More specifically, the invention relates to methods to treat or prevent neurological or psychiatric conditions mediated by astrocytes.
INTRODUCTION
[0004] Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
[0005] After the discovery of glial cells, and the subsequent definition of the different classes of glia, the structure of astrocytes provided the first clues to their function in the nervous system. The processes of the astrocyte are contained within a distance of about lOOμm and processes of the same cell contact neuronal membranes and the vasculature, where they form endfeet around the endothelia and smooth muscle. On the neuronal side, the astrocyte makes contact with synapses in several regions of the brain in a structure that has been defined as a tripartite synapse: the astrocytic process is associated with the pre and postsynaptic elements of the synapse. Because the astrocyte can contact the vasculature and synapses, it has been proposed that one function of this cell is to provide metabolic support to the neuron, a function
, that is probably important in supporting neuronal function during elevated levels of neuronal activity. Similarly, this structural relation has led to the idea of a reciprocal arrangement in which activity in the synapse is transmitted through signaling cascades of the astrocyte to the vasculature to locally regulate blood flow to provide metabolic support tuned to the demand of the level of neuronal activity.
[0006] At the synaptic level, it has been estimated that the processes of one astrocyte make contact with over 100,000 synapses. This synaptic relation serves many functions which include the clearance of synaptic transmitters. When a synaptic transmitter is released from the presynaptic nerve terminal to act on postsynaptic receptors, it is important that it is quickly cleared from the synapse in order to prevent receptor desensitization to maintain the fidelity of synaptic transmission.
[0007] Upon activation, astrocytes release chemical transmitters. The release of these gliotransmitters, in a process termed gliotransmission, can lead to paracrine actions on astrocytes supporting inter-astrocytic Ca signals, as well as signaling to neurons to regulate neuronal excitability and synaptic transmission. Astrocytes have been shown to release several transmitters, including glutamate, D-serine, ATP, adenosine, homocysteic acid, taurine and peptides such as atrial natriuretic factor (ANF).
[0008] Glutamate, D-serine, ATP, and adenosine, four gliotransmitters, have all been demonstrated to exert synaptic modulatory actions. Glutamate can have presynaptic effects mediated by either metabotropic glutamate receptors (mGluRs) or by kainite receptors that induce an enhancement of transmitter release. D-serine regulates NMDA receptor functions and synaptic plasticity. ATP can act through postsynaptic P2X receptors to induce an elevation of postsynaptic Ca+2 which is thought to drive the insertion of AMPA receptors to mediate an increase synaptic transmission. Additionally, and as a consequence of its extracellular hydrolysis, released ATP can cause a reduction in synaptic transmission mediated by presynaptic adenosine Al receptors, and modify neuronal excitability through the activation of GIRK (or Ikir) which hyperpolarizes the neuronal membrane potential.
[0009] Recently, it has been shown that astrocyte-dependent regulation of synapses is mediated through adenosine, and that this gliotransmitter allows the astrocyte to act as an intermediary in signaling between networks of synapses. It is well known that there is a tonic level of extracellular adenosine in the brain which causes a presynaptic inhibition of transmitter release from certain excitatory synapses. Additionally, adenosine can accumulate in an activity- dependent manner. Activity in presynaptic neurons leads to the activation of astrocytic signaling cascades which in turn cause the release of the gliotransmitter ATP, which is hydrolyzed to
adenosine causing an enhanced inhibition of synaptic transmission. Moreover, when adjacent synaptic pathways are studied, a brief high frequency stimulation of just one of these pathways leads to the activation of the astrocyte which in turn causes adenosine accumulation and the depression of the non-stimulated pathway. Thus, by controlling the level of extracellular adenosine, the astrocyte is coordinating the strength of networks of synaptic connections.
[0010] As many of the synapses in the CNS are tripartite in nature, disruption of astrocytic supportive functions and/or disruption of gliotransmission will consequently lead to disruption in synaptic transmission, synaptic plasticity and neuronal excitability. On a systems level, this translates to a disruption of brain physiology and behavioral abnormalities. Because of the privileged access of astrocytes to neurons and synapses these glial cells exert powerful regulation of neuronal elements in the brain.
[0011] In animal studies, immunostaining has been used as an index of the state of the astrocyte. Using antibodies directed against an astrocyte specific protein glial fibrillary acidic protein (GFAP), brain insults such as brain trauma or status epilepticus, have been shown to lead to astrocytic hypertrophy (reactive astrocytosis) and proliferation.
[0012] Injury to the nervous system caused by traumatic head injury, stroke or status epilepticus can lead to the delayed death of neurons in the limbic system and later to epilepsy (Lemos T et al. (1995) Exp. Brain Res. 102:423-428). Temporal lobe epilepsy (TLE), the most prevalent form of epilepsy in humans, is induced by insults to the nervous system and may not manifest itself until months or even years following injury in humans, weeks or months in rodents. TLE is considered a symptomatic epilepsy in which a prior insult stimulates, through a period of epilepto genesis, re-organizations of cellular structure and function that underlie epilepsy.
[0013] Status epilepticus induces a variety of alterations in the nervous system occurring at distinct periods following the onset of status epilepticus. During status epilepticus per se, GABA receptors are internalized. Later, after the termination of status epilepticus, during a latent period in which behaviorally detected seizures are not occurring, the so-called latent period of epileptogenesis, new GABA receptors with a different sub-unit composition are expressed ( Otis TS et al. (1994) Proc. Natl. Acad. Sci. USA 91:7698-7702; Brooks-Kayal AR et al. (1998) Nat. Med. 4:1166-72; Nusser Z et αl. (1998) Nature 395:172-7; Brooks-Kayal AR et al. (1999) J. Neurosci. 19:8312-8; Buhl EH et α/.(1996) Science 271:369-73; Gibbs JW et al. (1997) J. Neurophysiol. 77:2139-52). Similarly, during this latent period there is a period of delayed neuronal death, birth of new neurons in the dentate gyrus, collateral sprouting, synaptogenesis and the appearance of reactive astrocytes (Sutula T et al. (1989) Ann. Neurol.
PMf T / 1 P Bϊ'ft iB > p -r~ifi":::ι
26:321-30; Houser ClC (ϊ 9912) Epilepsy Res. Suppl 7:223-34; Houser CR (1999) Adv. Neurol 79:743-61; Houser CR et al. (1996) Epilepsy Res. 26:207-18; Houser CR et al. (1992) Epilepsy Res. Suppl. 9: 41-8; de Lanerolle NC et al. (1989) Brain Res. 495:387-95; Babb TL et al. (1984) Epilepsia 25:729-40; Babb TL et α/.(1991) Neuroscience 42:351-63; Fonseca CG et al. (2002) Brain Res. 929:105-16; Garzillo CL et al (2002) Epilepsia 43 Suppl 5:107-9). Finally, the culmination of these and other likely events lead to the generation of the epileptic brain in which spontaneous recurrent seizures develop. Following status epilepticus there is a latent period, during which no further seizures occur, followed weeks later by the onset of spontaneous seizures (epilepsy). During the latent period many pyramidal neurons die, days after status epilepticus (Mohajeri MH et al. (2004) Genes Brain Behav. 3:228-39; Mazarati A et «/.(2004) Neuroscience 128:431-41; Borges K et al. (2003) Exp. Neurol. 182:21-34), and cell and molecular reorganizations within the nervous system lead to the generation of epilepsy (epileptogenesis). The current therapeutic strategy is to treat epileptic patients with anticonvulsants. There is currently no treatment following the initial insult that will prevent epileptogenesis.
[0014] One percent of the population has a seizure disorder and ten percent of the population will have a seizure during its lifetime. In some patients, seizure symptoms can be alleviated or reduced in frequency by the lifelong use of anti-convulsants. However, relatively little headway has been made in understanding the mechanisms that lead to the generation of epilepsy following an episode of status epilepticus or after other types of injury to the nervous system. For example, following a traumatic head injury, there is no known treatment that will prevent epileptogenesis even though we know that up to 50% of individuals with moderate to severe traumatic brain injury are highly likely to develop epilepsy.
[0015] In addition to epilepsy, various neurodegenerative diseases are known to involve alterations of astrocytes, including frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (FormanMS et al. (2005) J. Neurosci. 25:3539-50). Reactive astrocytosis can also play a supportive role following neuronal injury. For example, ablation of astrocytes in mice with spinal cord injuries demonstrated failure of the blood brain barrier, demyelination, neuronal death, and motor impairment compared with parallel injured mice in which astrocytes were maintained (Faulkner JR et al. (2004) J. Neurosci. 24:2143-55).
[0016] Disruption of astrocytic support can also lead to excitotoxic damage. For example, knockout mice which do not express GLT-I, a major astrocytic glutamate transporter, develop seizures and pathology in the hippocampus. These phenotypes together with direct experimental evidence have led to the hypothesis that reduced expression of transporters in the
motor cortex and spinal cord can contribute to the degeneration of neurons m amyotrophic lateral sclerosis (ALS).
[0017] Gliotransmission may play a role in schizophrenia. Though schizophrenia is a complex disorder in which abnormalities in dopamine signaling have been strongly implicated, the recent discovery of the importance of NMDA receptor function is a breakthrough discovery. Treatment of human subjects with phenycyclidine (PCP), an NMDA receptor antagonist, induces a broad range of schizophrenic-like symptomatology including both positive and negative symptoms. Furthermore, PCP administration in rodents results in effects reminiscent of human schizophrenia such as working memory deficits, motor problems, and social behavior abnormalities. These initial results have led to the formulation of the glutamate hypothesis of schizophrenia. This hypothesis states that a prefrontal cortex glutamate deficiency is responsible for schizophrenia symptomatology. Such a deficiency in glutamate results in NMDA receptor hypofunction at prefrontal synapses which may contribute directly to the negative symptoms of schizophrenia. By means of corticostriatal projections, prefrontal NMDA receptor hypofunction results in increased dopamine levels in the striatum. Striatal dopamine contributes directly to the positive symptoms of schizophrenia. Additional support for the glutamate hypothesis comes from the fact that cycloserine, a partial agonist of the glycine-binding site on the NMDA receptor (also the D-serine binding site), is used in the treatment of schizophrenia. Clinical trials evaluating D-serine in ameliorating symptoms of schizophrenia are ongoing.
[0018] For the various conditions described above, among many others, there is a need for better therapies that inhibit or treat their onset or progression, and that do so with minimal negative side effects to the patient, and with fewer side effects than current therapies.
SUMMARY OF THE INVENTION
[0019] The invention features methods for treating or preventing neurological or psychiatric conditions in a subject. Various conditions can be treated or prevented using the inventive methods, and these conditions include, without limitation epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP-17, Hepatic encephalopathy, or Lupus encephalitis.
[0020] In one aspect, the methods comprise modulating the expression or activity of one or more proteins that participate in calcium signaling within astrocytes. Such proteins include, but are not limited to mGluR receptors, Gq, phospholipase C, Regulator of G Protein Signaling (RGS), IP3 phosphatase, IP3 Receptor, P2Y1, P2Y2, P2Y4, PIP2 kinase, PIP2
phosphatase, and caibmdm. Calcium signaling is targeted to modulate gliotransmission oi various chemical transmitters such as, but not limited to, glutamate, adenosine, ATP, and D- serine.
[0021] Modulating the expression or activity of the various proteins that participate in calcium signaling can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins. Various agonists and antagonists can be used to modulate the expression or activity of the proteins. Exemplary antagonists include, without limitation, MPEP, MTEP, SIB-1893. SEB-1757, GPant-2A, PPADS, and U-73122.
[0022] hi one aspect, the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of glutamate. Non-limiting examples of such proteins are SNARE proteins, synaptotagmin IV, Munc-18, VGLUTl, VGLUT2, or VGLUT3. Modulating the expression or activity of the various proteins that participate in the expression or release of glutamate can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins. Clostridial toxins and antagonists are exemplary ways to modulate the proteins that participate in the expression or release of glutamate.
[0023] In another aspect the methods comprise modulating the expression or activity of at least one protein that is activated by glutamate. Non-limiting examples of such proteins include NMDA receptors and AMPA receptors, and preferred targets include the NR2B subunit of the NMDA receptors. Modulating the expression or activity of the various proteins that are activated by glutamate can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins. Non-limiting examples of suitable antagonists are is D- AP5, (R)-AP5, PBPD, EAB515, LY233536, MK-801, Memantine, Ketamine, L-701,324, L- 689,560, GV196771A, Ro 25-6981, Co 101949, and Ifenprodil.
[0024] In another aspect, the methods comprise modulating the oscillation of free cellular calcium, particularly in astrocytes. One preferred means to modulate the oscillation of free cellular calcium is administration of a calcium chelator. Non-limiting examples of calcium chelators suitable for use in the present invention include EDTA, EGTA, diazo-2, FURA-2, Di- Bromo-BAPTA, and BAPTA-AM.
[0025] m another aspect, the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of ATP or adenosine. Non-
... .|ϊ" iu is ./" ilirβ-iii ci)^ iir.1HT-. d; rut. rέ , π. T A T,p , . , , « . . , ™ τ, r limiting examples of such proteins include SNARE proteins, bradylαnin receptors, mGluR5,
P2Y1, P2Y2, P2Y4 and noradrenergic receptors. Modulating the expression or activity of the various proteins that participate in the expression or release of ATP or adenosine can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins. Clostridial toxins and agonists are exemplary ways to modulate the proteins that participate in the expression or release of ATP or adenosine. Exemplary agonists include, but are not limited to, OAG, DAG lipase inhibitor RHC-80267, glutamate, ATP, norepinephrine, acetylcholine, and bradykinin. Modulation can also be by means of inhibiting diacylglycerol kinase or diacylglycerol lipase.
[0026] In another aspect, the methods comprise modulating the expression or activity of one or more proteins that participate in the expression or release of D-serine. One non-limiting example of such proteins is the NMDA receptor. Modulating the expression or activity of the various proteins that participate in the expression or release of D-serine can be carried out at the genetic or protein level, such as by targeting the genes encoding the proteins with antisense oligonucleotides, RNAi, or siRNA, or by use of an agonist or antagonist to the proteins. Exemplary agonists suitable for use in the present invention include, but are not limited to, ACBD, homoquinolinic acid, D-serine, cycloserine, D-cycloserine, glutamate, ATP, norepinephrine, acetylcholine, and bradykinin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 shows cell-wide Ca+2 oscillations in astrocytes lead to elevated glial glutamate release, excitation of neurons and delayed neuronal death. A) hi normal animals- afferent activity, in addition to its synaptic actions, causes local Ca+2 signals that remain restricted to the processes of astrocytes. Following status epilepticus these same inputs stimulate an astrocytic Ca+2 signal that propagates to the cell-body (Bl) which in turn invades all processes of the cell (B2). Cell- wide Ca+2 elevations evoke the release of large quantities of glutamate that synchronously depolarize groups of pyramidal neurons (B3). This enhancement of gliotransmission stimulates delayed neuronal death and re-organization underling epileptogenesis.
[0028] Figure 2 shows that application of Fluo-4AM to the surface of the cortex leads to the loading of astrocytes with the Ca+2 indicator fluo-4 which is confirmed by co-loading with sulforhodarnine 101. Images were acquired -lOOum beneath the cortical surface in vivo.
[0029] Figure 3 snows that injection into cortex with πuo-4AM (green) predominantly loads neurons, with weak labeling of astrocytes. However, subsequent surface labeling with SRlOl allows bright labeling of astrocytes (red).
[0030] Figure 4 shows two photon photolysis and imaging in vivo. Caged nitric oxide was photoreleased onto cortical astrocytes labeled with the Ca 2 indicator fiuo-4 (pseudocolor display). Images shown before (left) following (middle, right) photolysis (right; scale 20um).
[0031] Figure 5 shows that status epilepticus evokes a persistent increase in astrocytic Ca+2 signals lasting for three days. A) 3D reconstruction of astrocytes labeled with SRlOl. B)-C) Time courses of somatic Ca+2 oscillation from normal mice and mice 3 days after SE. Each cell was imaged from different mice. D) Frequency distribution of ΔF/F0 from mice showing enhanced Ca+2 signaling following SE (day 3 and 7 are each significantly different from control; p < 0.01; Kolmogorov-Smirnov test). E) Time course of change in Ca+2 oscillations reported as the integral of the ΔF/Fo signal (p < 0.002; n value is between 4 and 8 animals for each time point). F) The probability that an individual cell will exhibit Ca+2 oscillations is unchanged by SE. G) Synchronization of oscillation between astrocytes. Ca+2 oscillation of two pairs of astrocytes are shown. H) Cross correlation analysis of oscillation of cell pairs shown in G demonstrates synchronization. I) Histogram showing that the peak cross-correlation is enhanced following SE. J) The first image shows the raw fluorescence image of two cells which have synchronized oscillations as shown in adjacent ΔF/Fo images.
[0032] Figure 6 shows cell-wide and local astrocytic calcium oscillations in vivo. A) Astrocytes from mice 3 days after SE exhibit cell-wide Ca+2 oscillations. A) An example of astrocyte (green cell labeled with Fluo-4) showing cell-wide oscillations invaded from process 1 (Al) and process 2 (A2) to cell body. B) An example of astrocyte from a control mouse showing local oscillations of process 3, which did not invade to cell body. C-D) Calcium oscillations of astrocyte shown in (A). Oscillations in cell body and processes are highly synchronized as shown in the time course of ΔF/F0. Boxes Al and A2 correspond to the images Al and A2 in A). P1-P3 representing the different processes. D) High temporal resolution measurements of normalized ΔF/Fo from box Al in (C) clearly showing the oscillation originates in the process. E) Cross correlations of soma and process 1 (Pl). F) Ca+2 oscillations from astrocyte shown in (B) demonstrate that oscillations in processes are limited in a local microdomain. Boxed region corresponds to the ΔF/FO images in B). P1-P3 represent the different processes. G) Cross correlations of soma and process 1 (Pl) showing no correlation in Ca+2 oscillations. H) Comparison of correlation coefficients at zero time-lag (* p<0.0025; n = 4 to 5).
stimulates astrocytic Ca+2 signal propagation in vivo. A) Sequential ΔF/F0 images showing that cortical astrocytes exhibit propagating Ca waves in vivo when stimulated by the niGluR5 agonist CHPG (ImM). B) Time courses of somatic Ca+2 fluorescence changes (ΔF/F0; examples of four cells in each case) in control animals the absence and presence CHPG (ImM) as well as with CHPG in the presence of MPEP (30μM) and ifenprodil (lOμM). Note that the mGluR5 antagonist MPEP and not the NR2B NMDA receptor antagonist, ifenprodil, attenuate CHPG-induced Ca+2 signaling. The boxed region corresponds to the images in A). C) Ca+2 signals are stimulated by DHPG (25μM) and CHPG (ImM). The mGluR5 antagonist MPEP, but not the NR2B NMDA receptor antagonist ifenprodil, significantly attenuate CHPG-induced Ca+2 signals (N = 4-8 animals per group). D) Examples of cross correlation analysis of Ca+2 oscillations between adjacent astrocytes with and without CHPG. E) Comparison of cross correlation coefficients at zero lag time in the absence and in the presence CHPG showing that this niGluR5 agonist stimulates coordinated Ca signals in groups of astrocytes (N = 5-6 animals per group). * p<0.05; **ρ<0.02; ***p<0.01.
[0034] Figure 8 shows astrocytic Ca+2 oscillation in vivo in mice three days post-SE is inhibited by mGluR5 antagonist MPEP. A) Fluo-4 labeled astrocytes before (left) and after (right) the mouse was co-injected with MPEP and rhodamine-dextran through the tail vein. B) Calcium oscillations of a cell pair were inhibited after MPEP injection. C) Calcium oscillations before and after acute administration of MPEP (lmg/kg tail vein injection), depicted as integration of ΔF/Fo. Also shown is the inhibition by four daily i.p. administrations of MPEP (20mg/kg). Note that the NR2B NMDA receptor antagonist ifenprodil (20μM) does not affect astrocytic Ca+2 signals. * p < 0.002 (N = 3 to 4 animals in each group).
[0035] Figure 9 shows that gliotransmission but not intra-cortical synaptic transmission activates the NR2B-containing NMDA receptors of layer 2-3 pyramidal neurons. A) Whole-cell recording from a pyramidal neuron showing SICs induced by bath application of CHPG (0.5 mM). In this as well as in panels B-E, experiments are performed in the continuous presence of TTX (1 μM). B) Left: Percentage of neurons showing increased SIC frequency after application of DHPG (10-20μM, 4 of 11 cells), CHPG (0.5-1 mM, 7 of 20) and CHPG and MPEP (0.5-1 mM and 50 μM respectively, 1 of 17). In the latter experiments, the slice was preincubated with MPEP for 5 minutes before the application of CHPG and MPEP. The ability of MPEP to block actions of CHPG was tested using Fisher's exact test (p<0.05). Right: Average frequency of SICs before and after DHPG (N = 4) and CHPG (N = 7) in the responsive neurons shown in the left panel. In this as well as in the other panels of this figure: * p< 0.05; ** p < 0.01. C) Average frequency of SICs under control conditions, in the presence of D- AP 5 (50 μM) and after D-AP5
was PhouCtT m/ 6 cUorSticqal& neu/roBns4 s-3nowOin3g Q STlrC ac +ti-vi .ty. S QτInCs were s tti.mulated by application of
CHPG (0.5-1 mM) or low Ca+2 containing ACSF. D-E) Mean amplitude of SICs under control conditions, in the presence of ifenprodil (D, 3 μM) or NVP-AAM077 (E, 0.4 μM) and after drug washout. Data are normalized to the amplitude of SICs recorded under control conditions. See inset for representative SICs under the different experimental conditions from the same cell. Number of averaged SICs is 37, 48 and 70 from 9 cells for panel D and 27, 25, 13 from 7 cells for panel E respectively. F-G) Representative experiments showing the time course of the NMDA ueEPSC-amplitude at basal condition (0 Mg+2 containing saline in the presence of NBQX 10 μM), during ifenprodil (F, 3 μM) and NVP-AAM077 (G, 0.4 μM) application and after drug washout. Subsequent application of DAP5 (50 μM) completely blocked the EPSC (not shown). Inset: average of 10 EPSCs in the different experimental conditions. EPSC were evoked by positioning the stimulating electrode intracortically, 100-150 μm from the recording pipette. H) Ifenprodil (left, 3 μM) does not decrease the average amplitude of the NMDA EPSC (N = 6 cells) while NVPAAM077 (right, 0.4 μM) drastically reduces its amplitude (N = 4 cells). Data are normalized to the average NMDA EPSC amplitude under control conditions.
[0036] Figure. 10 shows MPEP and ifenprodil protect neurons from status epilepticus- evoked death. FJB stained sections of murine cortex from control (A) and mice three days following SE, (B) show FJB-labeling in layer 2/3, the region in which in vivo imaging and slice electrophysiology was performed. (C) Time course of FJB labeling. (D) The region identified by the dashed lines in (B) is shown from animals treated with glutamate receptor antagonists, as labeled. (E) MPEP (20mg/kg), ifenprodil (20mg/kg), and MK-801(lmg/kg) significantly reduce the number of FJB-labeled cells three days following SE, while NVP-AAM077 (2mg/kg) is not protective. (F) In parallel experiments the number of NeuN labeled neurons in cortical layers 2/3 seven days following SE were counted using an automated system. Similar to results obtained with FJB, MPEP and ifenprodil, but not NVP-AAM077, protected neurons from delayed death that normally follows SE. * p<0.05, ** p < 0.010; N= 4-7 animals in each condition.
[0037] Figure 11 shows photolytic elevation of Ca+2 in an astrocyte-evokes a slow inward current in CAl pyramidal neurons once the Ca+2 elevation in this glial cell confronts the neuronal dendrite (white overlay). Addition of the NMDA receptor antagonist, D-AP5 blocks this glial induced neuronal current. The NR2B-selective antagonist ifenprodil was applied to slices and showed a selective reduction of SIC amplitude with little effect on synaptic NMDA receptors (Fellin et al, 2004). This demonstrates that glial glutamate acts on extrasynaptic NMDA receptors. Additionally, activation of NR2B-containing NMDA receptors can activate a neuronal cell death pathway.
„ . , > . -,
[0058] Figure 12 snows injection of adenovirus containing GFAP promoter into rodent cortex directs the expression of receptor-EGFP fusion construct in astrocytes (individual cells not by arrows) Calibration 50 μm.
[0039] Figure 13 shows astrocyte-specific expression of the dn-SNARE domain. (A) Two lines of animals were generated, hGFAP.tTA and tetO.SNARE. When these lines are crossed, Dox suppresses SNARE, EGFP, and lacZ expression. (B) β-galactosidase (β-Gal) expression on a parasagittal section of dn-SNARE mouse brain (scale bar, 1 mm). (C and D) Slices from dn-SNARE mice (±Dox) show transgene regulation by Dox as reported by β-Gal (scale bar, 200 μm). (E) EGFP and β-Gal fluorescence and (F) GFAP immunoreactivity demonstrate expression of gene products in astrocytes. (G) Merged (E) and (F) images. (H to J) In hippocampal CAl, (H) EGFP and β-Gal transgenes are not expressed in neurons identified by (I) NeuN immunoreactivity. (J) Merged (H) and (I) images.
[0040] Figure 14 shows astrocytes regulate synaptic transmission and modulate plasticity through the control of extracellular adenosine. (A and B) Slope of Schaffer collateral CAl. fEPSP slope was larger in slices from dn-SNARE mice expressing this transgene (-Dox, n = 10 mice) compared to dn-SNARE controls (+Dox, n = 9 mice) and wild-type (wt) littermates (+Dox, n = 9 mice; -Dox, n = 10 mice). **, P < 0.02; scale bars, 1 mV, 10 ms. Stim, the dashed line represents the stimulus voltage (Stim) used to evoke the individual example traces shown as inserts. (C and D) The magnitude of theta-burst LTP was smaller when dn-SNARE was expressed in astrocytes (dn-SNARE - Dox, n =10 mice; dn-SNARE + Dox, n = 9 mice; wt + Dox, n = 9 mice; and wt - Dox, n = 10 mice). *, P < 0.05. (E) DPCPX (800 nM) augmented the slope of the fEPSP in slices from wild-type (**, P < 0.05) but not dn-SNARE (-Dox) mice. (F to H) The amplitude of theta-burst LTP in slices from wild-type animals was (F) blocked by D-AP5 (50 mM, n = 4 slices), (G) essentially unchanged by ifenprodil (10 μM, n = 9 slices) and (H) reduced to an extent similar to that observed in dn-SNARE mice (-Dox) by incubation in DPCPX (800 nM, n 0 6 mice). Data are presented as mean ± SEM.
[0041] Figure 15 shows dn-SNARE expression in astrocytes reduces extracellular ATP, a source of adenosine that regulates synaptic transmission. (A and B) CCPA (10 nM) reverses the effects of dn-SNARE expression by (A) reducing the fEPSP slope and (B) augmenting theta- burst-induced LTP (n = 7 slices; scale bar, 1 mV, 10 ms). (C) The ectonucleotidase inhibitor ARL67156 (50 μM) reduces fEPSP slope in wild-type slices (n = 3 slices), an action reversed by the P2 antagonist RB-2 (2 μM). ARL67156 does not change the fEPSP slope of dn-SNARE slices (n = 4 slices), demonstrating an absence of background ATP when dn-SNARE is expressed in astrocytes. (D) ATP, in the presence of RB-2 to block P2-mediated actions, reduces
^ _ ^ ^ ^ ^ fEPSP^slope in dl>SNARE' mice ^f=I slices), an action reversed by DPCPX (800 nM). Data are presented as mean ± SEM.
[0042] Figure 16 shows dn-SNARE expression in astrocytes prevents adenosine- mediated heterosynaptic depression. (A) Schematic representation of the experimental setup, showing two independent pathways Sl and S2. Stimulation of pathway Sl, 50 ms before S2 (top trace), does not change the amplitude of S2 fEPSP compared to stimulation of S2 alone (middle trace). The lower trace shows the top and middle traces superimposed. (B and C) A 100-Hz, 1-s tetanus delivered to pathway Sl evokes a depression of the S2 fEPSP (n = 4 mice). DPCPX (800 nM) (B) reduces heterosynaptic depression (n = 4 slices), which (C) is absent in slices from dn- SNARE (n = 5 mice) compared to wild-type slices (n = 6 mice). Data are presented as mean ± SEM.
[0043] Figure 17 shows that using vasculature as landmarks (A, B), which is loaded with rhodamine dextran, individual astrocytes can be identified, after removal of the animal from the imaging platform and after reattachment, the same identified astrocytes can be identified for subsequent studies. This approach can be used to study the impact of pharmacological antagonists/agonists on cell survival.
[0044] Figure 18 shows that transgenic mice which selectively express a dnSNARE transgene only in astrocytes (as defined in figures 13-16) to reduce the astrocyte-dependent accumulation of adenosine show a higher frequency of seizures in epileptic animals. Data are shown for three sequential analysis periods, in the presence and absence of doxycycline.
[0045] Figure 19 shows that treatment of the cortex with B APTA/AM, which selectively chelates Ca+2 in astrocytes, attenuates neuronal death as assayed by fluorojade B labeling. P<0.05
DETAILED DESCRIPTION
[0046] Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.
[0048] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or
±10%, "more preferably ±5°%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0049] As used herein, "epileptogenesis" refers to any molecular or cellular event that is part of the inception, development, transition, or progression of epilepsy.
[0050] The term "nervous system" refers to the entire nerve apparatus of an organism, and includes the central nervous system which comprises the brain and spinal cord, and the peripheral nervous system, which comprises the cranial and spinal nerves, autonomic ganglia, and plexuses, as well as the sympathetic and parasympathetic nervous systems.
[0051] A "neuron" or "neuronal cell" refers to any cell in the nervous system that conducts nerve impulses.
[0052] A "condition" is any state of being. This term is not limited to diseases or disorders, which can be considered an unhealthy state of being. A condition can be a transitional state of being. A condition can be a healthy or normal state of being. A subject having a condition need not manifest clinical symptoms.
[0053] "Psychiatric condition" refers to conditions of the mind.
[0054] "Neurological condition" refers to conditions of the nervous system.
[0055] A "convulsion" is an involuntary contraction or series of contractions of the voluntary muscles. Convulsions can, but need not accompany a seizure.
[0056] The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a protein.
[0057] The term "agonist" is used in the broadest sense, and includes any molecule that enhances, stimulates, or facilitates a biological activity of a protein.
[0058] "Test compound" refers to any purified molecule, substantially purified molecule, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material that can be analyzed using the methods of the present invention. Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Biomolecules include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized de novo. Test compounds can be defined in terms of structure or composition, or can be undefined. The compound can be an isolated product of unknown structure, a mixture of several known products, or an undefined composition comprising one or more compounds. Examples of undefined compositions include cell and tissue extracts, growth
»'" ft „ ft .->- V ■:;:■!' »...ι ..» " , . ι .. medium m which prokaryotic, eukaryotic, and archaebactenal cells have been cultured, fermentation broths, protein expression libraries, and the like.
[0059] The term "treating" refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluations.
[0060] The term "effective amount" refers to an amount of a biologically active compound or fragment, homolog, analog, or conjugate thereof sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without limitation, the amelioration of a neurological or psychiatric condition.
[0061] The terms "modulate" "modulating" or "modulation" refer to any change, including without limitation, any increase, decrease, enhancement, inhibition, suppression, or alteration, particularly with respect to the expression or activity of the proteins described herein, as well as the genes encoding said proteins.
[0062] The term "calcium oscillation" refers to any fluctuation in calcium concentration, especially as occurs in cellular signal transduction.
[0063] It has been discovered in accordance with the present invention that astrocytes promote neuronal cell death and dysregulation in a variety of neurological and psychiatric conditions, including epileptogenesis, convulsions, schizophrenia, and depression. It has further been discovered that astrocytes mediate these and other conditions through gliotransmission. Accordingly, in one aspect, the invention provides methods for treating or preventing a neurological or psychiatric condition in a subject in need of such treatment. Such methods comprise modulating the expression or activity of one or more proteins that participate in calcium signaling within astrocytes. The methods are effective for treating or preventing a neurological or psychiatric condition in which the calcium signaling mediates the expression or release of a chemical transmitter by the astrocyte and/or wherein the expression or release of the transmitter mediates, in whole or in part, the neurological or psychiatric condition in the subject. However, the inventive methods are not limited to conditions that are initiated, mediated, exacerbated, or sustained by the astrocyte itself. Because of the privileged access of astrocytes to neurons and their synapses, it is possible to change synaptic transmission and neuronal
. , .. e- t . . _. , i • , j. • Λ i - ■ excitability by modulating gliotransmission. Thus, when neurological conditions and psychiatric states have as a locus a change in synaptic transmission or neuronal excitability, targeting molecules to astrocytes will enable gliotransmission to act through the neuron and synapse to ameliorate the condition. That is, targeting calcium signaling in or gliotransmission of any of the various chemical transmitters expressed or released by the astrocyte can provide a therapeutic or preventative effect for conditions that are initiated, mediated, exacerbated, or sustained by the synapse, neuron, or any other cell in which gliotransmission by an astrocyte will produce an effect.
[0064] An astrocyte is a type of cell found in the brain and spinal cord. An astrocyte is a small, star-shaped glial cell (a cell that surrounds and supports nerve cells). The process by which astrocytes release chemical transmitters is referred to as gliotransmission. For example, Ca+2 signals in astrocytes evoke the release of glutamate and astrocytic glutamate acts on neuronal NR2B-containing NMDA receptors, which have been associated with cell death. It has not been known heretofore that injury to the nervous system, for example, as occurs in status epilepticus, induces enhanced Ca+2 signaling in astrocytes that lasts for days and that is correlated with a period of delayed neuronal death that is known to occur following this injury. It has also been heretofore unknown that neuronal death associated with epileptogenesis can be inhibited or even blocked by introducing agents that perturb this astrocyte to neuron signaling pathway.
[0065] Neuronal death is a pathology observed in multiple neurological tissue diseases. Such diseases include epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Huntington's disease, Parkinson's disease, and peripheral neuropathy, to name only a few. The methods of the invention have application to prolong neuronal cell survival in such neurodegenerative diseases. Moreover, the methods of the present invention are applicable to prolong neuronal cell survival in a subject that has suffered an insult, injury, trauma, or the like to the nervous system, especially an insult, injury, trauma, or the like to the brain. The injury to the nervous system may be a physical injury, particularly traumatic injuries, stroke, status epilepticus and the like. The survival of the neuronal cells is prolonged where cell death would be expected to occur, for example, at the inception, development, transition, or progression of neurodegenerative diseases. On preferred aspect of the invention utilizes methods to prolong neuronal cell survival for the treatment or prevention of epilepsy.
[0066] Various proteins that participate in calcium signaling in the astrocyte have been identified. These proteins include, without limitation, mGluR receptors such as niGluR5, GTP binding protein Gq, Regulator of G Protein Signaling (RGS) proteins, such as RGS2, IP3
phosphatase, IP3 receptors, P2Y receptors such as P2Y4, PIP2 kinase, PIP2 phosphatase, and calbindin. The modulation of the production of such proteins can be effectuated, for example, by increasing or decreasing expression of one or more of the proteins at the genetic level such as by increasing or decreasing the expression of the genes that encode such proteins.
[0067] Various methods for the regulation of gene expression are known in the art. Regulation of genes can occur, for example, at the transcription level, e.g., by inhibiting transcription. Regulation of genes can also occur, for example, at the post-transcription level, e.g., by inhibiting or degrading the mRNA. Antisense nucleic acids are one well-known approach to post-transcriptionally regulate gene expression. Full-length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be utilized. The use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art.
[0068] Small interfering RNAs (siRNA) can also be used, and such techniques are well-established in the art. RNA interference (RNAi) can also be used for post-transcriptional regulation of the proteins that participate in calcium signaling. A review of the RNAi technique is found in Marx, J. (2000) Science, 288:1370-1372. In brief, traditional methods of gene suppression, employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the corresponding protein. RNAi also operates on a post-translational level and is sequence specific, but suppresses gene expression far more efficiently. Exemplary methods for controlling or modifying gene expression are provided in WO 99/49029, WO 99/53050 and WOO/75164, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. In these methods, post-transcriptional gene silencing is brought about by a sequence-specific RNA degradation process which results in the rapid degradation of transcripts of sequence-related genes. Studies have shown that double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene constructs that produce transcripts with self- complementary regions are particularly efficient at gene silencing.
[0069] It has been demonstrated that one or more ribonucleases specifically bind to and cleave double-stranded RNA into short fragments. The ribonuclease(s) remains associated with these fragments, which in turn specifically bind to complementary mRNA, i.e., specifically bind to the transcribed mRNA strand for the gene of interest. The mRNA for the gene is also degraded by the ribonuclease(s) into short fragments, thereby obviating translation and expression of the gene. Additionally, an RNA-polymerase may act to facilitate the synthesis of
numerous copies of the short fragments, which exponentially increases the efficiency of the system. A unique feature of RNAi is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism.
[0070] Another available method for gene regulation in the present invention is the use of short hairpin RNAs (shRNA). A vector containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell by any means suitable in the art. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to siRNA molecules and are used by the cell to mediate RNA silencing of the desired protein.
[0071] Thus, available genetic information such as the nucleotide sequence, polypeptide sequence, etc. of the proteins that participate in calcium signaling or gliotransmission in the astrocyte can be used to generate gene silencing constructs and/or gene- specific self-complementary, double-stranded RNA sequences that can be delivered by conventional art-known methods. The sequences for the various proteins described herein are known and can be readily obtained through any of the various databases known and available to those of skill in the art.
[0072] A gene construct can be employed to express the self-complementary RNA sequences. Alternatively, cells can be contacted with gene-specific double-stranded RNA molecules, such that the RNA molecules are internalized into the cell cytoplasm to exert a gene silencing effect. The double-stranded RNA should have sufficient homology to the targeted gene to mediate RNAi without affecting expression of non-target genes. The double-stranded DNA is at least 20 nucleotides in length, and is preferably 21-23 nucleotides in length. Preferably, the double-stranded RNA corresponds specifically to a polynucleotide of the present invention. The use of small interfering RNA (siRNA) molecules of 21-23 nucleotides in length to suppress gene expression in mammalian cells is described in WO 01/75164. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.). See WO 01/68836. See also: Bernstein et al, RNA (2001) 7: 1509-1521; Bernstein et al, Nature (2001) 409:363-366; Billy et al, Proc. Nat'l Acad. Sci USA (2001) 98:14428-33; Caplan et al, Proc. Natl Acad. Sci USA (2001) 98:9742-7; Carthew et al, Curr. Opin. Cell Biol (2001) 13: 244-8; Elbashir et al, Nature (2001) 411: 494-498; Hammond et al, Science (2001) 293:1146-50; Hammond et al, Nat. Ref. Genet. (2001) 2:110-119; Hammond et al, Nature (2000) 404:293- 296; McCaffrrey et al, Nature (2002): 418-38-39; and McCaffrey et al, MoI. Ther. (2002) 5:676-684; Paddison et al, Genes Dev. (2002) 16:948-958; Paddison et al, Proc. Nat'l Acad. Sci
/ ,.. ,,, i . 1!" .::: ,, ~
USA (7002) 99:1443-48; Sui et al, Piroc. Natl Acad. Sci USA (2002) 99:5515-20. U.S. Patents of interest include U.S. Pat. Nos. 5,985,847 and 5,922,687. Also of interest is WO/11092. Additional references of interest include: Acsadi et al, New Biol. (January 1991) 3:71-81; Chang et al, J. Virol. (2001) 75:3469-3473; Hickman et al., Hum. Gen. Ther. (1994) 5:1477- 1483; Liu et al, Gene Ther. (1999) 6:1258-1266; Wolff et al, Science (1990) 247: 1465-1468; and Zhang et al, Hum. Gene Ther. (1999) 10:1735-1737: and Zhang et al, Gene Ther. (1999) 7:1344-1349.
[0073] Methods of the present invention include the use of gene therapy to regulate RNA interference and to over-express proteins. In such a strategy, for example, expression can be selective to neurological tissue, and more specifically, selective to astrocytes through the use of astrocyte-specific promoters. See, e.g., Wang CY et al (2006) Gene Ther. 2006 May 25; [Epub ahead of print]; Namikawa K et al (2006) Gene Ther. 2006 Apr 20; [Epub ahead of print]; and, Vandier D et al (2000) Cancer Gene Ther. 7:1120-6.
[0074] In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143- 4146 (1986)). The oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups.
[0075] There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, ex vivo, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral vectors and viral coat protein liposome mediated transfection (Dzau et al, 1993, Trends in Biotechnology, 11 :205-210). Viral vector mediated techniques may employ a variety of viruses in the construction of the construct for delivering the gene of interest. The type of viral vector used is dependent on a number of factors including immunogenicity and tissue tropism. Some non-
limiting examples oi viral vectors useful in gene therapy include retroviral vectors (see e.g., U.S. Patents 6,312,682, 6,235,522, 5,672,510 and 5,952,225, ), adenoviral (Ad) vectors (see e.g., U.S. Patents 6,482,616, 5,846,945 ), baculovirus vectors, and adeno-associated virus (AAV) vectors (see, e.g., U.S. Patents 6,566,119, 6,392,858, 6,468,524 and WO 99/61601 ). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, and the like. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis can be used for targeting and/or to facilitate uptake, e.g.,. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et ah, J. Biol. Chem., 262:4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87:3410-3414 (1990). For review of the currently known gene marking and gene therapy protocols see Anderson et ah, Science, 256:808-813 (1992).
[0076] hi the astrocyte, calcium signaling facilitates enzymatic pathways that proceed to induce the expression and/or release of various chemical transmitters by the astrocyte. Non- limiting examples of such chemical transmitters produced by astrocytes include glutamate, D- serine, and ATP, which is converted in the extracellular space to adenosine. The invention contemplates that its methods are broadly applicable to other chemical transmitters that are expressed or released as a result of the activation of calcium signaling, including such transmitters that are identified in the future. The expression or release of such chemical transmitters can mediate the expression and/or release of other chemical transmitters. For example, glutamate can induce the release of D-serine and ATP. ATP can induce the release of glutamate. ATP can induce the release of ATP. NO can induce the release of glutamate. PGE2 can induce the release of glutamate.
[0077] The modulation of the expression or activity such proteins that participate in calcium signaling in an astrocyte can also be accomplished by means administering at least one antagonist to the subject. In some aspects, the modulation of the expression or activity of such proteins that participate in calcium signaling in an astrocyte can also be accomplished by means administering at least one agonist to the subject. Antagonists or agonists are preferably administered in therapeutically effective amounts, which will be known to those of skill in the art, or could be readily determined by routine empirical testing using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to
_
, |H" IUu1If Λ" Λ4'^ IUf & X, E- ^" ."SiU .,"Φ . J? , - A A ■ j •, those skilled m trie art. The effective amount of the agonist or antagonist may depend on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the neurological or psychiatric condition. Preferably, a therapeutically effective dose of the antagonist or agonist will provide therapeutic benefit without causing substantial toxicity to the subject.
[0078] It is preferred, but is not critical, that the antagonist or agonist used is specific for a particular protein in the calcium signaling pathway, and non-specific antagonists or agonists that modulate the proteins can be used. Any agonist or antagonist for the calcium signaling pathway proteins in astrocytes that is now known in the art, or is later discovered is suitable for use in the methods of the present invention. Nonlimiting examples of antagonists useful in the methods of the present invention include: For mGluR5 receptors: 2-methyl-6-(phenylethynyl)- pyridine (MPEP); 3-[2-(methyl-l, 2-thiazol-4-yl)ethynyl pyridine (MTEP); [E]-2-Methyl-6-[2- phenylethenyljpyridine (SIB-1893); 6-Methyl-2-(ρhenylazo)-3-pyridinol (SIB-1757); for Gq: GpAntagonist-2A (GPant-2A); and, for phospholipase C: l-[6-[[17beta-3-methoxyestra- l,3,5(10)trien-17-yl]amino]hexyl]-lH- pyrrole-2,5- dione (U-73122), and 4-[[4-Formyl-5- hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-l,3-benzenedisulfonic acid (PPADS).
[0079] The expression or secretion of the proteins involved in Ca+2 signaling can also be inhibited at the translation level. For example, various agents can be used to target cellular protein expression machinery. For example, the inhibitor may specifically suppress expression of calcium signal pathway proteins from the ribosomes, or may target expressed proteins for proteolysis, such as by ubiquitination of the proteins.
[0080] Modulation of the expression of proteins that participate in calcium signaling in the astrocyte can also be by way of increasing expression of certain proteins in the pathway. For example, increased expression of proteins can be carried out through overexpression of known regulatory proteins such as RGS2 and 1,4,5-trisphosphate (IP3) phosphatase in the cell. RGS proteins are negative regulators of G protein-coupled signaling pathways, and the term "RGS" is deemed to encompass the over 20 RGS proteins that have been isolated to date, (see, De Vries L et al. (2000) Annu. Rev. Pharmacol. Toxicol. 40:235-71). IP3 phosphatase negatively regulates IP3-mediated calcium release. Over expression of such proteins can be accomplished by means of genetic engineering, as set forth above. For example, the genes encoding such proteins can be expressed under a strong promoter, a constitutive promoter, or an inducible promoter. The promoter can be tissue or cell specific, and can be specific for neurological tissues such as
astrocytes or glial "cells. "Examples' of strong promoters include the CMV promoter, SV40 early promoter, SV40 later promoter, SV2 promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, beta actin promoter, and other promoters shown effective for expression in eukaryotic cells. Nonlimiting examples of neurological tissue-specific promoters include the glial fibrillary acidic protein promoter (GFAP) (for astrocytes), and myelin basic protein promoter (for glial cells).
[0081] Proteins can also be used to modulate the expression or activity of proteins that participate in calcium signaling in astrocytes. For example, a polyclonal or monoclonal antibody that specifically binds to a protein that participates in calcium signaling in astrocytes such as a niGluR receptor, Gq, or phospholipase C can be used. In addition, any polypeptide that interacts with and/or modulates proteins that participate in calcium signaling in astrocytes can be used, e.g., a polypeptide that is identified using the presently described assays. Further, any compound that is found to or designed to interact with and/or modulate the activity or expression proteins that participate in calcium signaling in astrocytes can be used e.g., a compound that is identified using the presently described assays. Any of the above-described molecules can be used to increase or decrease the expression or activity of proteins that participate in calcium signaling in astrocytes, or to otherwise affect the properties and/or behavior of proteins that participate in calcium signaling in astrocytes, e.g., stability, intracellular localization, interactions with other intracellular or extracellular moieties, and the like.
[0082] The inventive methods are applicable to treat or prevent any neurological or psychiatric condition that is induced, mediated, exacerbated, or sustained, in whole or in part, by calcium signal pathways in astrocytes, whether the condition is induced, mediated, exacerbated, or sustained by the astrocyte itself, or by the synapse, neuron, or other cell in which gliotransmission by an astrocyte produces an effect. Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP-17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
[0083] Also featured in accordance with the present invention are methods for treating or preventing a neurological or psychiatric condition in a subject which comprise modulating the expression or activity of one or more proteins that participate in the expression or release of glutamate. The methods are particularly applicable where the expression or release of glutamate
initiates',' mediates, facilitates, exacerbates, or sustains, in whole or in part, the neurological or psychiatric condition in the subject. The modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
[0084] Various proteins that mediate glutamate release from astrocytes have been identified. These proteins include, without limitation, synaptotagmin IV, synaptobrevin II, Cellubrevin, SNAP-23, Synataxin I, Munc-18, VGLUTl, VGLUT2, and VGLUT3.
[0085] The modulation of the expression or activity of such proteins can be effectuated at the molecular level, including the nucleic acid or protein level, according to the methods described herein. The modulation of the expression or activity such proteins can also be accomplished by means of administering at least one antagonist or agonist to the subject. Preferably, such antagonists or agonists are specific for at least protein that mediates glutamate release from astrocytes, but specificity is not critical, i.e., nonspecific antagonists or agonists can also be used.
[0086] In one aspect, an agent administered to the subject inhibits the expression or activity of proteins that participate in glutamate expression or release by means of expression of the SNARE domain in order to prevent formation of the SNARE complex. The SNARE complex forms when four SNARE domains (soluble NSF attachment protein (SNAP) receptors), that are provided by three proteins come together to form a macromolecular protein complex. The proteins are comprised of a vesicular protein {e.g., synaptobrevin II) a plasma membrane protein {e.g., syntaxin) and a cytosolic protein {e.g., SNAP-25 in neurons, and SNAP-23 in astrocytes). Synaptobrevin and syntaxin each provide one SNARE domain, while SNAP-23 provides two. Expression of the cytosolic tail of the vesicle protein, which contains the SNARE domain, acts in a dominant negative manner to prevent the formation of the SNARE complex, thereby preventing the exocytosis of chemical transmitter {i.e., ATP) from the astrocyte.
[0087] The expression of a dominant negative form of the calcium-sensing protein synaptogamin TV can also be used. This dominant negative form of synaptotagmin TV consists of mutations to the C2B domain, a putative Ca+2-binding domain of Syt IV in which the first and the second aspartate residues of this domain were mutated to asparagines (D318,324N, referred to henceforth as B-D 1,2N), or the third aspartate and the fourth glutamate residues were mutated to asparagine and glutamine, respectively (D378N,E380Q, referred to henceforth as B- D3N,E4Q). Expression of these dominant negative molecules in astrocytes blocks the release of glutamate. (Zhang et al. (2004) Proc. Natl. Acad. Sci. USA. 101:9441-6)
10088] The expression or activity oi such proteins that participate in the expression or release of glutamate in an astrocyte can also be inhibited by administering to the subject a therapeutically effective amount of at least the enzymatic domain of at least one Clostridial toxin. It is contemplated that the entire toxin, or only the functional domains of the toxin, such as those that comprise the enzyme's active site, can be administered to the subject. Clostridial toxins, especially the botulinum toxin and tetanus toxin, are highly specific proteases that cleave SNARE proteins. Non-limiting examples of Clostridial toxins that can be administered in accordance with the present invention include the tetanus toxin, and botulinum toxins A, B, Cl, D, E, F, and G. The quantity of each respective toxin that would need to be administered to the subject, and the frequency of administration can be empirically determined according to any method that is suitable in the art.
[0089] Inhibition of the expression or activity of glutamate has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by glutamate expression or activity. Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP- 17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
[0090] Also featured in the present invention are methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of one or more proteins that are activated by glutamate. In some aspects, the methods are applicable where the activation of the proteins by glutamate initiates, mediates, facilitates, exacerbates, or sustains, in whole or in part, the neurological or psychiatric condition in the subject. Activation can occur following the gliotransmission of glutamate.
[0091] Various proteins that are activated by glial glutamate have been identified. Such proteins can be located in the synapse, proximal to the synapse, or even distal to the synapse. Such proteins outside of the synapse are generally referred to as extrasynaptic proteins. Non- limiting examples of proteins activated by glutamate include N-methyl-D-aspartate (NMDA) and α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) receptors. Glutamate can interact with any subunit on the NMDA receptor, including the NRI subunit, the NR2A subunit, or the NR2B subunit. Interference with the interaction of glutamate with the NR2B subunit is particularly preferred for purposes of the inventive methods.
[0092] Modulation of the expression or activity of proteins that are activated by glutamate can occur at the molecular level (nucleic acid or protein), as described herein. In some aspects, inhibition can be effectuated by means of administering at least one antagonist to the subject in a therapeutically effective amount. Nonlimiting examples of antagonists useful in the methods of the present invention include (i?)-2-ammo-5-phosphonopentanoate(D-AP5); (R)- AP5; (R)-CPP-ene; (±)-cώ-4-(4-phenylbenzoyl)piperazine-2,3-dicarboxylic acid (PBPD); (S)-Ά- amino-5-(phosphonomethyl)[l,r-biphenyl]-3-propanoic acid (EAB515); (±)-6-(lH-Tetrazol-5- yl-methyl) decahydroisoquinoline-3-carboxylic acid (LY233536); Dizocilpine/(5S,10R)-(+)-5- Methyl-10,1 l-dihydro-5Η-dibenzo[a,d]cyclohepten-5,10-imine (MK-801); 3,5- Dimethyladamantan- 1 -amine (Memantine); Ketamine; L-701,324; L-689,560; GV196771A; (+/- )-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-l-piperidine propanol (Ro 25- 6981); Co 101949; and Ifenprodil.
[0093] Modulation of the expression or activity of proteins activated by glutamate has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, exacerbated, or sustained, in whole or in part, by the activation of such proteins by glutamate in astrocytes or outside of astrocytes, such as in the synapse or extrasynaptic regions. The modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes. Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP- 17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
[0094] The invention also features methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the oscillation of free cellular calcium. In some aspects, the methods are particularly applicable where the oscillation of the calcium mediates, in whole or in part, the neurological or psychiatric condition in the subject. The modulation can be targeted to astrocytes, however, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes.
[0095] By way of example and not of limitation, modulating oscillation of cellular calcium can occur via diminishing the availability of free calcium in the astrocyte. The availability of cellular calcium can be suppressed, for example, by use of a calcium chelator. Calcium cheltors are known in the art, and include, without limitation, EDTA, EGTA, diazo-2,
FUΪIA-2,
and BAPTA-AM. Calcium chelators can be administered directly to the subject, for example, by injection into the cerebrospinal fluid of the ventricles of the brain, or onto the cerebral cortex. Calcium chelators can be injected in one bolus or could be continuously infused from a pump attached to a catheter, as is standard in medical practice. Because astrocytes are coupled in a syncitium by gap junctions, cells local to the injection site take up dye and then deliver it throughout the brain through this coupled network. This approach allows selective loading into astrocytes of BAPTA-AM, as well as other membrane permeant antagonists of chelating agents or AM coupled reagents.
[0096] Modulation of the oscillation of free cellular calcium has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by the oscillation of free cellular calcium. Non-limiting examples of such conditions include, epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, the supportive role for astrocytes in the repair of nervous tissues such as the spinal cord following injury, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HTV dementia, bipolar disorder, FTDP-17 (other tauopathies), Hepatic encephalopathy, Lupus encephalitis, and the like.
[0097] In considering the role of gliotransmission in disorders of the nervous system, it is intriguing that enhanced excitatory gliotransmission, for example, in terms of Ca+2 oscillations and glutamate release, contributes to epileptiform activity and seizures. On the flip side, it has been discovered that reduced NMDA receptor function contributes to schizophrenia. Since the astrocyte specific gliotransmitter D-serine stimulates NMDA receptor function, gliotransmission is a target to enhance the function of this receptor and overcome this psychiatric state. Thus, modulation of gliotransmission on one hand reduces the deleterious effects of glutamate on neuronal death and epileptogenesis, and on the other hand overcomes the negative symptoms of schizophrenia.
[0098] D-serine is selectively synthesized and released from astrocytes. Without intending to be limited to any particular theory or mechanism of action, it is believed that decreasing the gliotransmission of D-serine will result in hypofunction of NMDA receptors. Accordingly, the invention feature methods to enhance D-serine release, and methods to enhance NMDA receptor function to treat or prevent neurological or psychiatric conditions. In some aspects, the methods are particularly applicable where the conditions are induced, mediated, exacerbated, or sustained, in whole or in part, by the diminished expression or activity of D- serine, or the diminished expression or activity of NMDA receptors. The modulation can be
targeted to astrocytes, however, the methods are not limited to conditions mediated,- facilitated, exacerbated, or sustained by astrocytes. In general, the methods comprise modulating the expression or release, i.e., gliotransmission, of D-serine, or modulating the expression or activity of the NMDA receptor
[0099] The modulation of the expression or release of D-serine, or the modulation of the expression or activity of NMDA receptors can be effectuated at the molecular level, including the nucleic acid or protein level, according to the methods described and exemplified herein. It is preferred that the expression or release of D-serine, and/or the expression or activity of NMDA receptors is enhanced. The modulation of the gliotransmission of D-serine can also be facilitated by means of administering at least one agonist to a subject that has a neurological or psychiatric condition. Suitable agonists include, but are not limited to D-serine, cycloserine, and D-cycloserine. The release of D-serine itself can be stimulated by various chemicals that induce Ca+2 oscillations in the astrocytes including glutamate, ATP, norepinephrine (NE), acetylcholine (Ach), and Bradykinin.
[0100] Modulation of the expression or release of D-serine, and/or the expression or activity of the NMDA receptor has utility to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by the gliotransmission of D-serine. Schizophrenia is a non-limiting example of such a condition.
[0101] The modulation of the expression or activity of NMDA receptors can also be accomplished by means of administering at least one agonist to the subject. Preferably, such agonists are specific for the NMDA receptors, but specificity is not critical, i.e., nonspecific agonists can also be used. Non-limiting examples of NMDA receptor agonists include N- methyl-D-aspartate, l-aminocyclobutane-cis-l,3-dicarboxylic acid (ACBD), Homoquinolinic acid, and the like.
[0102] Using molecular genetic strategies that were applied selectively to the astrocyte, it has been discovered that adenosine is derived selectively from astrocytes. It has also been discovered that enhanced adenosine production can prevent seizures and convulsions. Thus, the invention also features methods for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of one or more proteins that participate in the expression or release of ATP and/or adenosine. In these methods, adenosine expression or release ameliorates, in whole or in part, the neurological or psychiatric condition in the subject. A primary source of adenosine is the hydrolysis of extracellular ATP that is released ATP, which is hydrolyzed to adenosine (Pascual O et al. (2005) Science 310:113-6). It has been
,. Mu a K 11 discovered m accordance with the present invention that adenosine accumulates secondarily to the release of ATP from astrocytes.
[0103] Various proteins have been identified as members of the pathways that facilitate adenosine expression or release. Examples of such proteins include, but are not limited to bradykinin receptors, mGluR5, P2Y1, P2Y2, or noradrenergic receptors. Without intending to be bound to any particular theory or mechanism of action, it is believed that any receptors that are coupled to the action of phospholipase C induce the release of ATP. In one aspect, the methods comprise enhancing the expression or activity of these proteins. In another aspect, the methods comprise enhancing the expression or release of ATP.
[0104] The methods can also comprise inhibiting the expression or activity of diacylglycerol kinase (DAGK) or diacylglycerol lipase. Diacylglycerol kinase converts diacylglycerol (DAG) to phosphatidic acid leading to an overall reduction in DAG levels. Similarly DAG lipase (DAGL) degrades DAG and inhibition of this enzyme leads to an increase of DAG levels and ATP release. Since released ATP is hydrolysed to adenosine, the inhibition of DAGK or DAGL will augment extracellular adenosine levels where it will have its natural anticonvulsant activity. DAGK inhibitors include, but are not limited to, 6-[2-(4-[(4- fluorophenyl)phenyl-methylene]-l-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-alpha] pyrimidine-5-one (R59022), and 3-(2-(4-[bis-(4-fluorophenyl)methylene]-l-piperidinyl)ethyl)- 2,3-dihydro -2-thioxo-4(lH)quinazolinone (R59949), among others.
[0105] Expression of the proteins that participate in the expression or release of ATP (which results in the accumulation of extracellular adenosine) can be enhanced at the molecular level, using, for example, the methods described herein, or by using any of the various means for enhancing gene or protein expression that are known and routinely practiced in the art. Expression can also be enhanced by means of administering a therapeutically effective amount of an agonist of the expression of such proteins. For example, it has been discovered that administration of the DAG analog, l-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates ATP release from astrocytes. Thus, OAG is one example of an agent that can be used with the inventive methods to facilitate ATP and thus, adenosine production. Additional agonists include, but are not limited to, DAG lipase inhibitor RHC-80267. The release of ATP itself may be stimulated by various chemicals that induce Ca+2 oscillations in the astrocytes including glutamate, ATP, NE, ACh, and Bradykinin. Facilitation of ATP and adenosine gliotransmission can also be achieved by inhibition of SNARE, as described and exemplified herein.
[0106] Such methods are applicable to treat or prevent any neurological or psychiatric condition that is induced, mediated, or sustained, in whole or in part, by diminished levels or the
absence of adenosine. Although modulation can be targeted to astrocytes, the methods are not limited to conditions mediated, facilitated, exacerbated, or sustained by astrocytes. Similarly, the methods are applicable to treat or prevent any such condition that can be ameliorated by adenosine. For example, and not by way of limitation, adenosine has anticonvulsive properties (Lee KS et al. (1984) Brain Res. 321:160-4.) Accordingly, one preferred condition that can be treated or prevented using the methods of the invention is convulsions. Sleep abnormalities, anxiety, hypoxia and cerebral ischemia can also be ameliorated by enhancing adenosine release.
[0107] Another aspect of the invention features methods to identify agents that modulate gliotransmission in astrocytes comprising contacting a test compound with an astrocyte and determining an increase or decrease in gliotransmission by the astrocyte in the presence of the test compound relative to the level of gliotransmission by the astrocyte in the absence of the test compound. The test compound can be assessed at multiple concentrations, and under varying environmental conditions such as temperature, oxygen, humidity, and the like.
[0108] The assays can be carried out using freshly isolated astrocytes, or using astrocyte cell lines such as U373 MG, or C6 glioma. The effect of the test compound on gliotransmission can be carried out for any of the chemical transmitters produced by astrocytes, including without limitation, glutamate, adenosine, and D-serine, or any combination thereof. Determination of whether gliotransmission is increased or decreased in the presence of the test compound can be carried out using any means suitable in the art, which can vary depending on the needs of the investigator, or on which chemical transmitter(s) of interest is being evaluated. The assay used can be qualitative or quantitative. Those of skill in the art will know appropriate assays for determining gliotransmission of the various chemical transmitters, as well as how to fine tune such assays, all of which are routine in the art.
[0109] The invention also features methods for identifying compounds that modulate gliotransmission by astrocytes using a combination of an in vitro and in vivo screening assay. In one aspect, a test compound is first screened in vitro as described herein, and then screened further in vivo to determine if the compound can modulate gliotransmission in the body. After the test compounds are administered to a subject, test samples are periodically taken from the subject and screened to determine if the test compound increased or decreased gliotransmission in neurological tissues within the subject. Once the test sample is taken from the subject, ex vivo screening on the test sample can be practiced according to the details described herein.
[0110] The invention also features methods to identify agents that prolong neuronal cell survival. In one aspect such methods are applied to identify agents that inhibit epileptogenesis in a subject. The methods comprise inducing status epilepticus in a test animal,
. administering a test compound to trie test animal, and determining whether the test compound inhibits epileptogenesis in the animal relative to the frequency and severity of epileptogenesis in animals not treated with the test compound.
[0111] Status epilepticus can be induced in a test animal according to any means that are suitable in the art. One preferred means is to administer pilocarpine to the test animal in an amount sufficient to evoke status epilepticus in the particular animal. The test animal can be any mammal, for example, mice, rats, rabbits, cats, dogs, non-human primates, guinea pigs, cows, horses, pigs, and the like. Preferably, the test animal is a mouse. The amount of pilocarpine sufficient to evoke status epilepticus can vary depending on the type, sex, age, or weight of the animal, and may require empirical determination. Such empirical determinations are routine in the art.
[0112] After induction of status epilepticus, the seizure is allowed to proceed for a period of time lasting for several minutes to several hours. The seizure can be allowed to proceed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, or more hours. Preferably, the seizure is allowed to proceed for 1 hour. After the seizure proceeds for the desired period of time, the seizure is preferably terminated. The seizure can be terminated according to any means that are suitable in the art. For example, the seizure can be terminated by administering to the animal diazepam in an amount suitable to terminate the seizure. The amount of diazepam sufficient to terminate the seizure can vary depending on the type, sex, age, or weight of the animal, and may require empirical determination, which are routine in the art.
[0113] After termination of the seizure, a test compound is administered to the animal. The test compound can be administered to the animal according to any means suitable in the art, such as by injection (intravenous, intramuscular, intradermally, and the like) orally, through inhalation, transdermally, enterically, and the like. The route of administration may vary according to the nature of the compound to be tested. The administration of the test compound can be intermittent or at a gradual, continuous, constant, or controlled rate. In addition, the time of day and the number of times per day that the test compound is administered can vary. Similarly, the number of days in which the test compound is administered may vary. The test compound may be administered over a period of weeks, months, or years.
[0114] Following the administration of the test compound to the test animal, it is to be determined whether the test compound affects neuronal cell survival. Although it is expected that the screening methods of the invention will identify compounds that enhance as well as diminish neuronal cell survival, those compounds that prolong cell survival subsequent to status epilepticus are desired. Determination of whether the test compound prolongs cell survival can
, be accomplished by screening neurological tissue for gliotransmission, particularly for glutamate, for calcium oscillation, or to determine whether glutamate binds the NR2B subuiiit of the NMDA receptor.
[0115] Calcium oscillation can be measured according to any means suitable in the art. Non-limiting examples of such means include two photon calcium imaging and voltage clamping, as exemplified herein. Methods of measuring glutamate are known in the art and can include, for example, the use of glutamate biosensors or fluorometric methods (Neurosci Methods. 1991 Mar; 37(1):7-14. Neuroreport. 1997 May 27;8(8):2019-23). Methods for determining receptor binding are known in the art and are thus not described herein in detail. AU such measurements may be taken on any neuronal tissue, for example, isolated astrocytes or glial cells, or from brain slice preparations, as described and exemplified herein.
[0116] The following examples are provided to illustrate the invention in greater detail. Examples 4, 6, 7, 9, 11, 13, 14, 16, 17, 18, and 19, below, are prophetic examples. AU of the examples herein are intended illustrate, not to limit, the invention.
Example 1
General Experimental Procedures
[0117] Astrocytes amplify glutamatergic synaptic activity. By applying in vivo two- photon imaging to cortical astrocytes, it has been that a one hour period of status epilepticus evokes an increase in the Ca+2 excitability of astrocytes that lasts for three days. Evidence demonstrates that the Ca+2 signaling behavior of the astrocyte changes from one in which Ca+2 oscillations are restricted to regions of the astrocytic processes (Figure IA) to a pattern in which they invade all processes of the cell; Ca+2 oscillations become a cell-wide property (Figure IB). Because the processes of one astrocyte contact more than 100,000 synapses and because the glial Ca+2 oscillations evoke the release of glutamate that acts on dendritic NMDA receptors, this change in behavior of the Ca+2 signal has the potential to amplify synaptic inputs to a region of an astrocyte by releasing glutamate to hundreds of thousands of dendritic locales. Following status epilepticus, the astrocyte amplifies local synaptic inputs through a cell-wide Ca+2 oscillation which stimulates glial glutamate to be released. This release acts on neuronal NR2B- containing NMDA receptors that are known to activate cell death pathways, thereby stimulating the synchronous excitation of pyramidal neurons, excitotoxicity (Figure IB) and, ultimately, epileptogenesis.
,,.,, ;; . , .;; [0118] P/zofo Imaging of Neurons and Astrocytes. An m vivo two-photon imaging approach that permits the study of Ca+2 signaling in identified astrocytes and neurons has been developed. Success in this approach has required the development of selective loading techniques that permit the introduction of fluorescent Ca+2 indicators into astrocytes and/or neurons. Two reproducible methods are available to achieve this result. First, Nimmerjahn et al (Nirnmerjahn et al., 2004) have demonstrated a simple method to load cortical astrocytes with small molecular weight Ca+2 indicators. After making a cranial window into the cortex, small molecular weight compounds such as fluo4-AM or sulforhodamine 101 are topically applied to the cortical surface for 30-45 min. This loading technique results in the loading of astrocytes with these dyes in essentially 100% of animals and even in adult animals (>1 year of age). Surface loading selectively loads Ca+2 indicators into astrocytes, never into neurons (Figure 2).
[0119] To load cortical neurons with Ca+2 indicator the AM ester of the dye is injected into the cortex as described (see, Stosiek et al., 2003). Membrane-permeant dye (e.g., fluo- 4AM) is ejected into the cortex, and 30-45 min later, neurons fluoresce and exhibit Ca+2 signals. Astrocytes can also load with the Ca+2 indicator. However, the astrocytes typically exhibit relatively weak fluorescence intensity with this loading method. Without being limited to any particular theory or mechanism of action, it is believed that this low fluorescence is attributed to the diffusion of the dye from these locally loaded cells out into the astrocytic syncitium through gap junction connections. In order to load both neurons and astrocytes with Ca+2 indicator, fluo- 4AM is injected into the cortex to label neurons, and then fluo-4AM and sulforhodamine are applied to the cortical surface to label the astrocytes. Neurons are loaded with Ca+2 indicator only while astrocytes are co-loaded with fluo-4 and sulforhodamine 101 (Figure 3).
[0120] A two-photon microscope can be used to study astrocyte-neuron interactions. This two-photon microscope integrates two scan control mechanisms, one to control the imaging and the other the photolysis pathway. The two-photon microscope is coupled to two Ti:Sappliire lasers, one of which is tuned to 720nm for photolysis, and the other of which is tuned to 820nm to excite fiuo-4 for imaging. Figure 4 shows the changes in Ca+2 level in an astrocyte and demonstrates that photo-release of nitric oxide (NO) from caged NO induces a Ca+2 elevation, which results from the influx of Ca+2 (Li et al. , 2003). This capability can be used to integrate two-photon photolysis with imaging to study the ability of the astrocyte to release glutamate and activate NR2B-containing NMDA receptors.
[0121] For in vivo two-photon Ca+2 imaging, FVB/NJ mice were anesthetized with urethane (1.5-2.0 mg/g body weight) and held in an immobilization device. Next, a circular cranial window (2.0 mm diameter) was drilled in the skull overlying the cortex. A metal frame,
to attach the skull to the microscope platform, was attached to the skull using cyanoacrylate, and the dura was carefully removed. 2μl of 50 μg fluo-4 AM mixed with 5 μl pluronic acid (20% pluronic acid plus 80% DMSO) was mixed with 30 μl ACSF (containing (in mM) 120 NaCl, 10 Hepes, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose; with pH 7.4) and applied through the cranial window to the surface of the cortex. After 45-60 min excess dye was removed by irrigation with ACSF. This protocol led to the selective labeling of astrocytes with the Ca+2 indicator, fluo-4.
[0122] Selectivity of labeling was confirmed using sulforhodamine 101. 100 μl of 100 μM SRlOl dissolved in ACSF was applied on the surface of the cranial window for 1-5 min before being washed away with ACSF. After 45-60 minutes, astrocytes were labeled selectively with SRlOl . A glass coverslip was glued over the cranial window on the metal frame and the gap between glass and cranial window is filled with 2% agarose pre-melted in ACSF solution. Mice were placed on a heating pad to keep them warmed at 37°C. In vivo two-photon imaging was performed using a Prairie Technologies Inc., Ultima scanhead attached to an Olympus BX51 microscope equipped with a 6Ox water immersion objective. A Chameleon Ti:Sapphire laser (Coherent, CA) was used for two-photon excitation, and a Mai-Tai Ti:Sapphire (Spectra Physics) laser for photolysis. Fluo-4 fluorescence was excited at 820 nm and emission was detected by an external photomultiplier tube while photolysis was performed at 720nm.
[0123] In some experiments 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was co- injected together with the fluorescent dye rhodamine-dextran through the tail vein. A mixture of 200 μl ACSF containing 10 mg/ml Rh-Dextran and MPEP to yield a final concentration of MPEP of lμg/g was injected into the tail vein. Successful injection was confirmed by the rapid appearance of rhodamine in the vasculature within the cortex. 30 minutes were allowed to elapse prior to re-commencing Ca+2 imaging to ensure that the MPEP had crossed the blood brain barrier. To study acute effects of ifenprodil and DHPG or CHPG on the Ca+2 oscillations, these drugs applied to the cortical surface for 30 minutes before re-commencing imaging. The cranial window as then re-filled with 2% agarose containing the same concentration of drug.
[0124] The fluorescent signals were quantified by measuring the mean pixel intensities of the region of interest using Metamorph software (Universal Imaging Corp). Ca+2 changes were expressed as ΔF/F0 values, where F0 was the baseline fluorescence. To express the magnitude of Ca+2 signals without subjective selection of threshold values, the ΔF/F0 signal was integrated over a 300s imaging period using Origin software.
[0125] Whisker-evoked potentials (EP) were recorded using a glass electrode filled with ACSF and connected to a Swam HC amplifier (Celica, Slovenia). Mouse whiskers were
stimulated with a 100ms air puff through a glass tube while EP was being recorded. Data were acquired with a Digidata 1320 interface and pClamp software (Axon Instruments, CA). Brain Slice Preparation and Patch Clamp Aecordings and Analysis
[0126] Transverse brain slices (300-400 μm) were prepared from FVB/NJ mice (Jackson lab, Bar Harbor, Maine) at postnatal days 12-45 as described previously (Pasti et ah, 1997). After cutting, slices were incubated at 37° C for a recovery period of lhr. The solution for slice cutting and incubation was (in mM): NaCl 120, KCl 3.2, NaH2PO4 1, NaHCO3 26, MgCl2 2, CaCl2 1, glucose 2.8, Na-pyruvate 2 and ascorbic acid 0.6 at pH 7.4 with O2 95 %, CO2 5%. In the recording chamber slices were continuously perfused with normal ACSF (in mM): NaCl 120, KCl 3.2, NaH2PO4 1, NaHCO3 26, MgCl2 1, CaCl2 2, glucose 2.8, at pH 7.4 with O2 95 %, CO2 5%. Low Ca+2 solution was obtained by replacing CaCl2 with EGTA (0.25 mM). Pipette resistance was 3-4 MΩ. Intra-pipette solution was (in mM): K-Gluconate 145, MgCl22, EGTA 5, Na2ATP 2, NaGTP 0.2, HEPES 10 to pH 7.2 with KOH. Patch clamp recordings were performed using standard procedures using MultiClamp-700B amplifiers (Axon instruments, Union city, CA) or 2400 patch-clamp amplifiers (A-M Systems Inc., Sequim, WA). Data were filtered at 1 KHz and sampled at 5 KHz with a Digidata 1320 interface and pClamp software (Axon Instruments). Experiments were performed at 30-35°C. Neurons were voltage-clamped at -60 mV. Evoked postsynaptic currents (EPSCs) were elicited by intracortical stimulation (0.1 Hz) with a bipolar tungsten electrode placed 100-150 μm from the recording pipette. The rise time of the NMDA component of the EPSCs and SICs was calculated with the 20-80% criterion and the decay time as the time constant of a single exponential fit. Data analysis and fitting were performed with Clampfit 9.2 (Axon Instruments), Origin (Microcal Software, Northampton, MA) and SigmaPlot 8.0 (SPSS Inc., Chicago, IL) software.
[0127] Induction of Status Epilepticus. Status epilepticus was induced in the 6-8 week- old male FVB/NJ strain of mice available from Jackson labs (Mohajeri et ah, 2004). Pilocarpine was the preferred agent for chemical induction of status epilepticus. Behavioral seizures were assessed according to the scale of Racine (Racine, 1972).
[0128] Thirty minutes prior to the injection of pilocarpine, methyl scopolamine (a muscarinic antagonist) was administered subcutaneously (s.c.) (1 mg/kg) to reduce adverse, peripheral affects. Status epilepticus was induced through s.c. injection of 350 mg/kg pilocaφine-hydrochloride, a muscarinic agonist. Animal behavior and seizure activity was documented throughout the procedure. One hour after the onset of status epilepticus, seizure activity was reduced by the administration of diazepam (5mg/kg) subcutaneously. Control mice (two groups) were injected either with saline or with a l/10th dose of pilocarpine. For
expeπments m which gliotransmission following status epilepticus is pharmacologically manipulated, MPEP and ifenprodil (0.2-20mg/kg) were administered intraperitoneally (i.p.) to mice together with diazepam. Antagonists were subsequently administered i.p. once daily for up to 3 days before euthanization.
[0129] Transcardial Perfusion and Immunocytochemistrγ Staining. Mice were anesthetized with halothane, and transcardially perfused first with ice cold phosphate buffered saline (PBS) and then 4% paraformaldehyde in PBS (pH=7.4). After perfusion, the brain was post-fixed in 4% paraformaldehyde PBS at 40C for 30 min. To prevent ice crystal formation, the brain was transferred to 30% sucrose and stored for several hours. Coronal sections of the brain (20 μm) were cut and collected serially on pre-cleaned slides, and were stored at -8O0C until use. For Fluoro-Jade B staining, brain sections were washed with PBS 3 times and then immersed into 0.0001% Fluoro-Jade B in 0.1% acetic acid solution at 40C for 1 hr. For double staining, sections were stained with either ApopTag kit (UpState) or biotinylated mouse anti-NeuN (1 : 100) followed by Fluoro-Jade B staining procedure as described above. All sections were counterstained with Dapi (1 : 1000, in PBS) and mounted using anti-fade mounting medium. Stained sections were viewed with epifluorescence and double-stained sections were examined using an Olympus fluoview 1000 confocal microscope and analyzed using Metamorph (Universal Imaging Corp). Four random sections were selected from every animal. The number of FJB-labeled cells was counted bilaterally by a blind investigator. To count the number of neurons in area C A3 and cortex cells images of DAPI and NeuN staining in area CA3 of the hippocampus and 70-150 μm beneath the surface of cortex were acquired with a digital camera. Automated software was used to identify and count neuronal cell bodies based on the co- localization of NeuN with DAPI. Manual inspection confirmed the accuracy of this automated approach., and analyzed.
[0130] Statistical Analyses. N values reported in in vivo studies represent the number of animals. In the in vivo imaging experiments Ca+2 measurements were generally made from 6- 10 astrocytes per animal, and the data were averaged to obtain a single value per animal. Similarly, in neuron and FJB counting studies, measurements were made on at least four sections which were used to obtain a single value representative of that animal. When multiple comparisons were performed ANOVA tests were performed and differences between individual groups were determined using the Newman-Keuls post- hoc tests. Electrophysiological studies were tested for significance using the student's t-test unless stated otherwise. Statistical significance of data was reached at p<0.05.
Example 2
Gliotransmission Mediates NMDA-2B Receptor-Dependent Neuronal Death Following
Status Epileptictts
[0131] Status Epilepticus triggers delayed astrocytic Ca+2 oscillations in vivo during the latent period of epileptogenesis. Chemically-induced epileptiform activity in brain slice preparations, or in vivo, in addition to evoking intense neuronal discharges stimulates oscillations of astrocytic Ca+2 raising the potential for the excitability of the astrocyte to be altered in a long- term manner that outlasts epileptiform (in vitro), or seizure activity (in vivo). This hypothesis was tested using two photon imaging of astrocytic Ca+2 (Nimmerjahn et al., 2004) to determine whether seizures per se persistently modify the excitability of astrocytes. Layer 2/3 astrocytes in the barrel cortex were selectively loaded with the Ca+2 indicator Fluo-4 two or more days after pilocarpine-induced status epilepticus (Fig 5A). In control animals (vehicle and sub-threshold pilocarpine injected; Fig. 5B), Ca+2 oscillations recorded at the astrocytic cell body were infrequent (N = 8), although present in about 60% of all cells studied (Fig. 5F). In contrast, astrocytic Ca+2 oscillations were elevated in astrocytes from experimental animals two-three days following status epilepticus (Fig. 5C-E: 2days post SE, N = 4 animals, p<0.05; 3days post SE, N =8 animals , P<0.00005). Ca+2 oscillations were synchronous between adjacent astrocytes (Fig. 5G- J) with large correlationcoefficients at zero lag time (Fig. 5H, I).
[0132] Delayed Ca+2 oscillations switch from local microdomain elevations to cell- wide signals. Bergmann glia and astrocytes exhibit a functional compartmentalization of intracellular signaling in brain slices in which Ca+2 oscillations are restricted to microdomains of a cell's processes (Grosche et al, 1999; Nett et al, 2002). In some preparations where movement of cells in vivo were absent the spatial pattern of Ca+2 oscillations was still able to be quantitatively analyzed, and was found to accurately reflect the qualitative behavior of signaling seen in the other preparations. Three days following status epilepticus, astrocytic Ca+2 signaling had changed from local elevations to a cell-wide oscillatory behavior (Fig. 6; n = 5 cells). Following status epilepticus, Ca oscillations originate within individual processes and propagate to the cell body and on to additional processes of the cell (Fig. 6 A, C-E). In contrast, in control animals, astrocytes exhibit compartmentalization of signaling (n = 4 cells). Ca+2 oscillations originate in a process and remain restricted to this microdomain (Fig. 6B, F-H). Ratiometric Ca+2 measurements, which control for movement artifacts, confirm these assertions (not shown). Thus, status epilepticus changes Ca+2 signaling from microdomain oscillations restricted to regions of processes to a signal that propagates through the cell entirety.
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[0133] niGluRj mediates enhanced astrocytic calcium oscillations following status epilepticus. Because niGluR5 contributes to astrocytic Ca+2 oscillations in brain slices, whether this receptor mediates the enhanced astrocytic Ca+2 signaling detected following status epilepticus was investigated. First, it was determined whether mGluR5 agonists would mimic in control mice the enhanced Ca+2 excitability detected following status epilepticus. Application of either 3,5-Dihydroxyphenylglycine (DHPG; 25 μM), a class I mGluR agonist, or 2-chloro-5- hydroxyphenylglycine (CHPG; 1 mM), a selective mGluR5 agonist, induced enhanced Ca+2 signaling within astrocytes (Fig. 7). Ca+2 oscillations which initiate within a cell's processes propagate through the entirety of the cell and couple to adjacent astrocytes in the form of a Ca+ wave (Fig. 7 A, B; N = 5 animals). As shown following status epilepticus, CHPG5 caused synchronized Ca+2 oscillations as detected by a large cross-correlation between the Ca+2 signals of adjacent astrocytes at zero lag time (Fig. 7D, E; N = 5 animals; p < 0.05). CHPG-induced Ca+2 signals are due to the selective activation of mGluR5 because they are prevented by the mGluR5 antagonist MPEP (30μM; Fig. 7B,C). After recording oscillations within astrocytic cell bodies of mice subjected to status epilepticus three days earlier (Fig. 8 A, B) the mGluR5 antagonist MPEP (lmg/kg weight) was injected through the tail vein, together with rhodamine- dextran as a positive label for successful injection. MPEP, which crosses the blood brain barrier, significantly reduced the astrocytic Ca+2 oscillations (Fig. 8B, C; N = 4 animals; P < 0.002). Taken together with the ability of mGluR5 agonists to induce astrocytic Ca+2 oscillations (Fig. 7), these results demonstrate the importance of mGluR5 for the activation of Ca+2 oscillations in astrocytes following status epilepticus.
[0134] mGluR5 -dependent activation of astrocytic Ca+2 signaling evokes NR2B- dependent NMDA receptor-mediated neuronal currents. Because of the potential for astrocytic Ca+2 oscillations to cause glutamate-mediated neuronal excitation, as has been described for thalamic and hippocampal pyramidal neurons (Angulo et at, 2004; Fellin et ah, 2004; Perea and Araque, 2005), studies in acutely isolated brain slices were performed to determine whether layer 2/3 cortical astrocytes excite cortical pyramidal neurons. Astrocytic Ca+2 oscillations evoked slow inward currents (SICs) mediated by NMDA receptors that can be identified based on four properties: their slow kinetics, insensitivity to TTX and NBQX, and their blockade by D-AP5. The class I mGluR agonist DHPG (10-20 μM) as well as the mGluR.5 selective agonist CHPG (0.5-1 mM) induced TTX insensitive SICs in layer 2/3 cortical neurons that were blocked by the mGluR5 antagonist MPEP (50 μM; Fig. 9 A, B; n =17, p<0.05). These slow currents share kinetics typical of previously identified SICs (Fellin et ah, 2004), which are one order of magnitude slower than NMDA-mediated synaptic-evoked currents (Table 1). In addition to
exhibiting slow Kinetics ana an msensitivity to TTX, SICs fulfill the remaining pharmacological criteria to assign their origin to the astrocyte: they are mediated by the selective activation of NMDARs since they are reversibly blocked by DAP5 (50 μM, Figure 9C) while NBQX (10 μM) does not change the amplitude and kinetics of SICs (Tablel). These results are consistent with mGluR5 inducing astrocytic Ca+2 oscillations (Figs. 7, 8) that cause glial glutamate release which in turn stimulates NMDA receptor-dependent SICs in cortical pyramidal neurons (Fig. 9).
Table 1.
SICs are significantly slower than NMDA receptor mediated EPSCx and SICs are independent of AMPA receptor activation. Mean rise, decay time, and amplitude for SICs and intra-cortical- evoked NMDAR EPSCs.
[0135] Because of the potential for astrocytic and synaptic glutamate to access distinct NMDA receptors (Fellin et ah, 2004) the relative sensitivity of SICs and intra-cortical evoked EPSCs to NVP-AAM077 (0.4μM) and Ifenprodil (3μM), which preferentially inhibit NR2A/C/D and NR2B-containing NMDA receptors, respectively, was evaluated. The amplitude of SICs were reversibly reduced in amplitude by ifenprodil (p < 0.01), while they were insensitive to NVP-AAM077 (Fig. 9D, E). In contrast, intra-cortical NMDA receptor-mediated EPSCs were reversibly attenuated by NVP-AAM077 (Fig. 9G-H; p < 0.01) but insensitive to Ifenprodil (Fig. 9F,H). The selective attenuation of synaptic NMDA receptors by NVP-AMM077 and attenuation of SICs by ifenprodil show that gliotransmission, that is mediated by SICs, selectively accesses extrasynaptic NR2B-containing NMDA receptors. Because NR2A but not NR2C or NR2D containing NMDA receptors exhibit rapid kinetics similar to the synaptic NMDA currents, it is likely that the NVP-AAM077 sensitive synaptic NMDA currents are mediated predominantly by NR2A-containing NMDA receptors. Thus, astrocytic, but not
, ,. , ...... .....,„ -. synaptic, glutamate selectively accesses extrasynaptic NR2B subunit-containing NMDA receptors.
[0136] Delayed neuronal death is suppressed by antagonists of gliotransmission but not synaptic transmission. Because mGluR5 stimulates the release of glial glutamate that activates NR2B subunit-containing NMDA receptors (Fig. 9), an NMDA receptor pathway implicated in mediating neuronal death (Hardingham et al, 2002), whether there is a linkage between gliotransmission and the delayed cell death that is known to follow status epilepticus was evaluated. It was first confirmed that the relative selectivity of pharmacological agents by monitoring astrocytic Ca+2 signals and whisker-deflection evoked potentials, m agreement with acute administration of MPEP (Fig. 8), daily intraperitoneal (i.p.) administration of MPEP (20 mg/kg), that commenced immediately following the termination of status epilepticus, reduced astrocytic Ca+2 oscillations (N = 3 animals, p <0.0025; Fig. 8C). In contrast, administration of MPEP had no impact on whisker-evoked potentials (N = 4 animals). Ca+2-dependent astrocytic glutamate acts on extrasynaptic NR2B-containing NMDA receptors (Fig. 9). Accordingly, acute cortical administration of ifenprodil (20 μM), an NR2B-selective antagonist, neither affected the whisker-evoked potentials (N = 3 animals) nor astrocytic Ca+2 oscillations (Fig. 7C, 8C; N = 3-4 animals), while as a positive control for drug access, TTX blocked evoked potentials (N = 3 animals, p < 0.0002). Thus, MPEP and ifenprodil can be used selectively to prevent astrocytic Ca+2 oscillations and to attenuate astrocyte-evoked SICs without significant actions on either synaptic NMDAR currents or on whisker-evoked synaptic potentials and neuron-based integration.
[0137] The time course of neuronal death that follows status epilepticus was determined by labeling sections from animals with fluorojade B (FJB) (Schmued and Hopkins, 2000), a fluorescent label that selectively identifies dying cells. Following status epilepticus, FJB-labeled neurons, which co-label with the neuronal marker NeuN, were observed in layer 2/3 of the cortex (Fig. 1OA, B), the same cortical region in which in vivo imaging and slice electrophysiology was performed. The peak of FJB-labeled neurons correlated with the period of astrocytic Ca+2 oscillations with labeling declining thereafter (Fig. 10C). Because of this temporal correlation the relative roles for gliotransmission and synaptic transmission in mediating this neuronal death was evaluated. One hour after the cessation of status epilepticus, four daily i.p. injections of MPEP (20 mg/kg, i.p.), ifenprodil (20 mg/kg, i.p.), MK801 (1 mg/kg, i.p.), NVPAAM077 (2 mg/kg, i.p.) or vehicle were commenced Administration of ifenprodil (N = 4 animals) or MPEP (N = 4 animals), antagonists that attenuate gliotransmission but have no actions on NMDAR-mediated synaptic transmission or whisker evoked field potentials, both
caused significant neuronal protection (Fig. 1OD, E; lfenprodil, p<0.01; MPEP, p<0.05). In contrast, NVPAAM077, which attenuates NMDAR-mediated synaptic transmission, did not provide neuronal protection (Fig. 1OD, E). MK801, a use-dependent NMDAR antagonist which does not select between NR2B- or NR2A-containing NMDA receptor subunits offered minor protection. As an alternative approach to FJB labeling, seven days were allowed to elapse following status epilepticus, after which NeuN-positive neuronal cell bodies were identified and counted. Similar to the FJB data, ifenprodil (N = 4 animals, p<0.05) and MPEP (N = 4 animals; p<0.05), but not NVP-AAM077 (N = 3 animals), afforded significant protection against delayed neuronal death that normally follows status epilepticus (Fig. 10F). hi agreement with these results in the cortex, a similar pattern of hippocampal pyramidal neuron protection by antagonists that selectively attenuate gliotransmission was also observed (not shown).
[0138] Additional support for an astrocytic origin of the SICs was provided by direct stimulation of the astrocyte. Focal UV photolysis was applied to single astrocytes that made contact with the dendrites of pyramidal neurons. When the Ca+2 elevation in the astrocyte approached the dendrite an SIC that was blocked by D-AP5 was detected (Figure. 11). Taken together, these results demonstrate that the Ca+2 oscillation of an astrocyte excites pyramidal neurons by the release of glutamate that acts in NMDA receptors. It is believed that astrocyte- evoked neuronal NMDA currents may be a generalized phenomenon of the nervous system as these events have been detected in each region of the nervous system that has been tested thus far, including areas CAl and C A3 of the hippocampus, cortex and nucleus accunibens.
[0139] Cell culture studies showed that astrocyte-derived glutamate can act on non- synaptic NMDA receptors (Araque et ah, 1998b). Brain slices were coupled with pharmacological approaches to distinguish between NMDA receptors containing different subunit compositions. The slow kinetics of the SIC raised the possibility that SICs may be mediated by NMDA receptors containing the NR2B subunit. To test this possibility, the NR2B- selective antagonist ifenprodil was applied to brain slices. These experiments showed a selective reduction of SIC amplitude with little effect on synaptic NMDA receptors (Fellin et ah, 2004). Since NR2B-containing receptors are known to be at extrasynaptic locales after synapses have developed (Tovar and Westbrook, 1999; Rumbaugh and Vicini, 1999) this important observation provides further support for the notion that glial glutamate acts on extrasynaptic NMDA receptors. Additionally, activation of NR2B-containing NMDA receptors can activate a neuronal cell death pathway.
Example 3
Antagonists that Act on the Astrocvte-Neuron Signaling Pathway Reduce Delayed Neuronal Death Following Status Epilepticus
[0140] It has been demonstrated that a period of enhanced astrocytic Ca+2 signaling correlates with a period of delayed neuronal excitotoxicity following status epilepticus. To determine whether this is a causal relationship, MPEP or ifenprodil were administered to mice intraperitoneally following status epilepticus. Without intending to be bound to any particular theory or mechanism of action, it is believed that, if the astrocytic Ca+2 signal evokes glutamate release that acts through NR2B-containing NMDA receptors, then administration of MPEP, to reduce Ca+2 oscillation frequency, or ifenprodil, to block the extrasynaptic NMDA receptor targets of glial glutamate, should reduce neuronal death.
[0141] Following the induction of status epilepticus, mice were administered either MPEP (20mg/kg) or ifenprodil (20mg/kg) i.p. on each of three days. Animals were then cardiac perfused, and brains were sectioned and stained with Fluoro-Jade B which labels dying neurons (Schmued and Hopkins, 2000). In parallel controls, Ca+2 oscillations in cortical astrocytes were imaged, confirming that they were at control levels following i.p. MPEP administration.
[0142] The number of Fluoro-Jade B-labeled cells in both cortex and area CA3 of the hippocampus was significantly reduced by either MPEP or ifenprodil as compared to vehicle controls. These results provide evidence that enhanced Ca+2 signaling in astrocytes stimulates neuronal death in vivo by releasing glutamate, which acts on NR2B-containing NMDA receptors.
Example 4
Regulator of GTP-Binding Protein Signaling 2 (RGS2) Expression in Astrocytes Uncouples
Receptor Activation from Ca+2 Signaling
[0143] To test of the role of gliotransmission in the regulation of delayed excitotoxicity and epilepto genesis will require the use of astrocyte-specific expression of transgenes to perturb this signaling pathway. Two complementary approaches will be developed to achieve this objective, hi the first approach, viral expression of RGS 2 will be used to block Ca+2 signaling in astrocytes, hi the second approach, astrocyte-specific inducible transgenic animals will be used.
[0144] RGS proteins regulate GTP binding signaling by terminating the active G protein signal (Hains et al, 2004). Receptor occupancy by an agonist causes the exchange of GDP for GTP and dissociation of Gα.CTp from the β/γsubunits. Gα.Gτp and βγ act on effectors to stimulate signaling. Termination of the signal is regulated by the GTPase activity of Ga which is controlled by RGS proteins. Overexpression of RGS proteins leads to an abrupt termination of the receptor activated signal. RGS2 interacts with Gqα the α-subunit that is predominantly
responsible for mediating the activation of phospholipase C, and thus the generation OfIP3 and Ca+2 release from internal stores, by a variety of receptors including niGluR5 and purinergic receptors such as P2Y1 and P2Y2.
[0145] The expression in astrocytes of RGS2 can perturb receptor-induced PLC and IP3-dependent Ca+2 signaling. RGS2 can be expressed together with DSRed2 in astrocytes and assayed ATP-induced Ca+2 signaling. In agreement with other studies RGS2 potently blocks Gq signaling pathways as ATP, even at concentrations as high as lOOμM (receptor KD ~ lμM), was unable to evoke Ca+2 signals compared to DsRed2 expressing cells. RGS2 has been investigationally introduced into adenoviral and lentiviral vectors that have been engineered to contain the GFAP promoter for astrocyte-specific expression of RGS2. Since it was demonstrated that transgenes can be selectively expressed in astrocytes in the cortex (Figure 12), this viral approach can be used to introduce RGS2 into astrocytes to determine whether this cell type-specific uncoupling of gliotransmission will reduce delayed excitotoxicity following status epilepticus.
Example 5 Astrocvte-Specific Inducible Transgenic Animals that Perturb Gliotransmission
[0146] The cytosolic portion of the SNARE domain of synaptobrevin 2 (amino acids 1 to 96) was selectively expressed in astrocytes, a manipulation that blocks gliotransmission. Two lines of transgenic mice were developed. In the first, GFAP.tTA, the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter drives the expression of the Btet-Off tetracycline transactivator (tTA). The second, tetO.SNARE, contains a tet operator (tetθ)-regulated SNARE domain and lacZ and enhanced green fluorescent protein (EGFP) reporter genes (Fig. 13A). Crossing lines yields mice in which SNARE, LacZ, and EGFP transgenes are expressed in GFAP-positive astrocytes, not in neurons (Fig. 13B and E to J), and in which transgene expression is suppressed by doxycycline (Dox) (Fig. 13, C and D). These animals are referred to as dominant negative SNARE (dn-SNARE) mice.
[0147] It was determined if astrocyte-specific expression of the dn-SNARE domain affects synaptic transmission and plasticity by using acutely isolated hippocampal slices. The slope of Schaffer collateral-evoked field excitatory postsynaptic potentials (fEPSPs) was significantly larger (P < 0.02) in dn-SNARE mice expressing the transgene (n = 10 mice) compared to either dn-SNARE mice maintained on Dox to prevent transgene expression (n - 9 mice) or wild-type littermates (Fig. 14, A and B) with no change in the fiber volley. Thus, a SNARE-dependent process in astrocytes influences basal synaptic transmission.
[0148] To ask whether astrocytes also modulate synaptic plasticity, long-term potentiation (LTP) was evaluated. Theta-burst stimulation applied to wild-type brain slices potentiated fEPSP slope by 232 ± 18%. The magnitude of LTP was significantly less (P < 0.05) in mice expressing dii-SNARE (172 ± 11%, n = 10 mice) (Fig. 14, C and D). Because the magnitude of LTP in wild-type mice was unaffected by maintenance on Dox, and yet this treatment prevented the change in LTP magnitude observed in dn-SNARE mice, it is believed, without intending to be limited to any particular theory or mechanism of action, that astrocytes control the available range for synaptic plasticity, by regulating the strength of basal synaptic transmission.
[0149] The potential roles for three gliotransmitters known to be released from astrocytes was evaluated to determine whether they coordinately regulate baseline fEPSP slope and the magnitude of LTP. D-2-amino-5 phosphonopentanoate (D-AP5, 50 mM, n = 4 mice) to block N-methyl-D-aspartate (NMDA) receptors, the target of glial-derived D-serine and glutamate, did not change fEPSP slope (Fig. 14E) and blocked LTP (Fig. 14F). Because glial glutamate preferentially activates NR2B subunit-containing NMDA receptors, ifenprodil (10 mM, n = 9 slices), an NR2B subunit-containing NMDA receptor antagonist, was tested and revealed actions on neither the fEPSP slope (Fig. 14E) nor LTP (Fig. 14G). The P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (PPADS) (5OmM, n = 5 slices) and reactive blue-2 (RB-2) (2 mM, n = 4 slices) had little effect on fEPSP slope (Fig. 14E), suggesting that ATP does not directly activate P2 receptors to mediate astrocytic potentiation of fEPSP and depression of LTP.
[0150] Released ATP can be converted into adenosine by ectonucleotidases. Because there is a tonic level of extracellular adenosine that acts through the Al receptor to persistently suppress excitatory synaptic transmission, whether adenosine mediated the effects of astrocyte- specific dn-SNARE expression was evaluated. The Al receptor antagonist 8-cyclopentyl-l,3- dipropylxanthine (DPCPX) (800 nM) increased the fEPSP slope of wildtype slices by 67 ± 19% (n = 6 mice) (Fig. 14E) and decreased the amplitude of LTP (n = όslices) (Fig. 14H) to 146 ± 20%, values similar to those measured in hippocampal slices obtained from dn-SNARE mice. In contrast, DPCPX (800 nM) had no effect on baseline synaptic transmission in slices from dn- SNARE mice (3 ± 14%, n = 4 slices) (Fig. 14E). The Al agonist 2-chloroN6- cyclopentyladenosine (CCPA, 10 nM) reversed the actions of dn-SNARE expression by reducing the amplitude of the baseline fEPSP (n = 7 slices) (Fig. 15A) and augmenting the amplitude of LTP (n = 7slices) (Fig. 15B).
loss from the cytosol through equilabrative transporters and accumulation resulting from ectonucleotidase-dependent hydrolysis of released ATP. Blockade of equilabrative nucleoside transporter 1 (ENTl) with S- (4-nitrobenzyl)-6-thioinosine (NBMPR, 100 nM), a highly selective ENT-I antagonist, led to a further reduction in basal synaptic transmission, which is consistent with a role for ENT-I in the uptake rather than the release of adenosine. As such, the release of ATP from astrocytes as the potential source of adenosine accumulation was evaluated by using synaptic transmission as a sensitive assay for this nucleotide. Because P2 receptor antagonists do not change the magnitude of synaptic transmission (Fig. 14E), there is insufficient extracellular ATP under normal circumstances to activate synaptic P2 receptors. Whether the ectonucleotidase inhibitor ARL67156 (50 mM) would lead to an accumulation of ATP that would modulate baseline fEPSP amplitude was assessed. Superfusion of ARL67156 caused an inhibition of synaptic transmission (Fig. 15C), an action that was reversed by the P2 antagonist RB-2 (2 μM, n = 3 slices). If adenosine accumulation is mediated by the hydrolysis of ATP released from astrocytes, it would be predicted that the ARL67156-induced synaptic suppression would be absent in slices from dn-SNARE mice. Superfusion of ARL67156 did not significantly change (9 ± 22%) fEPSP slope in dn-SNARE slices, compared to a -56 ± 8% reduction in wild-type slices (Fig. 15C). Luciferin/luciferase imaging of extracellular ATP detected significantly less (P < 0.05) extracellular ATP in slices from dn-SNARE mice (12 ± 7 counts s"1, n = 4 mice) compared to wild-type (26 ± 8 counts s"1, n = 4 mice). These results are consistent with dn- SNARE blocking a previously described vesicular mode of ATP release from astrocytes. It was next assessed whether exogenous ATP would restore Al receptor-mediated, adenosine-induced synaptic suppression. In the presence of RB-2, to block direct actions of ATP on P2 receptors, application of exogenous ATP to dn-SNARE (n = 4 slices) or wild-type (n = 4 slices) slices similarly induced a reduction in fEPSP slope that was reversed by the Al antagonist DPCPX (Fig. 15D).
[0152] Tetanic stimulation of Schaffer collaterals causes an adenosine-mediated, heterosynaptic depression of neighboring unstimulated synapses. However, the source of the adenosine remains an enigma. Because stimulation of the Schaffer collaterals induces Ca signals in astrocytes, which in turn evoke the release of gliotransmitters, and because heterosynaptic suppression has been indirectly linked to a glial-dependent mechanism, the dn- SNARE mice were used to determine whether astrocytes mediate heterosynaptic depression. Two independent pathways (Sl and S2) were each stimulated at 30-s intervals to monitor baseline synaptic transmission (Fig. 16A). In wild-type slices, tetanic stimulation of pathway Sl
(100 Hz, 1 s) evoked homosynaptic LTP together with a heterosynaptic depression of the neighboring S2 pathway. The addition of an Al antagonist (DPCPX, 800 nM) prevented heterosynaptic depression (Fig. 16B). To control for effects of enhanced baseline transmission that result in the presence of DPCPX, normal artificial cerebrospinal fluid (ACSF) was switched to one containing 2.4 mM Ca+2 and 0.6 mM Mg+2, which enhanced synaptic transmission to 172 ± 8.9% (n = 3 slices) of that in control mice. Heterosynaptic depression was still observed to be intact (64.9 ± 8.2%, n = 3 slices) compared to ACSF controls (72.0 ± 8.6%, n = 3 slices). To determine whether astrocytes mediate adenosine-dependent depression, this study was repeated using dn-SNARE slices and found a virtual absence of heterosynaptic depression (Fig. 16C).
[0153] These studies place the astrocyte at center stage in the control of adenosine. Glialreleased ATP, which is rapidly hydrolyzed to adenosine, leads to a persistent synaptic suppression mediated by Al receptors. Because adenosine is implicated in the control of wake to-sleep transitions as well as responses to hypoxia, the identification of the central role of the astrocyte in regulating this nucleoside offers mechanistic insights into these processes.
[0154] The kinetics of ATP hydrolysis and adenosine accumulation provide a synaptic network with unique spatiotemporal conditions to control synaptic transmission. Fast-acting synaptic transmitters such as g-aminobutyric acid and glutamate have high-affinity uptake systems in the vicinity of the synapse that constrain the time and distance over which a transmitter acts. Synaptic activation of an astrocyte to release ATP removes these constraints, because it takes -200 ms before adenosine begins to accumulate. This provides time for ATP diffusion to distant sites, where it depresses synaptic transmission through accumulated adenosine, thereby providing a mechanism for cross-talk to distant synapses. In addition to activity-dependent actions, astrocytes, by persistently suppressing excitatory synaptic transmission, enhance the capability of synapses to express synaptic plasticity. Thus, the integration of synaptic activity by the astrocyte leads to a widespread coordination of synaptic networks. By suppressing excitatory transmission, astrocytes regulate the degree to which a synapse maybe plastic, and during the induction of LTP, astrocyte-derived adenosine depresses neighboring unstimulated pathways.
Example 6 Vasculature as Landmarks to Relocate Previously Identified Individual Astrocytes
[0155] During certain experiments, individual astrocytes may need to be relocated to determine whether the addition of agonists and antagonists affect their Ca+2 signaling. For this relocation, rhodamine dextran can be injected into the vasculature to identify specific volumes in
, „■• i i the brain, much like one uses a road. Figure 17 shows that with this approach, the same individual astrocytes can be identified and relocated such that astrocytes in different brain volumes can be imaged before a pharmacological agent is provided, and then afterwards to deteπnine the resultant changes in signaling.
Example 7
Determination of Whether Status Epilepticus Evokes Enhanced Ca+2 Oscillations of
Hippocampal Astrocytes
[0156] Preliminary studies have demonstrated that cortical astrocytes exhibit a period of enhanced excitability following status epilepticus. Thus, it must be determined whether similar changes in glial excitability are present in the hippocampus, a site of considerable delayed neuronal death and the source of later epileptiform activity. The mechanism of these changes in glial excitability can also be determined. Accompanying the status epilepticus- induced change in glial Ca+2 oscillations is a change in the spatial distribution of the Ca+2 signal. Ca+2 signals in astrocytes in control animals are generally restricted to microdomains within regions of processes whereas Ca+2 signals that originate from a process spread throughout the cell entirety following status epilepticus. Because preliminary studies suggest that the mGluR5 agonist DHPG permits cell-wide Ca+2 oscillations in control animals and MPEP reduces the frequency of such oscillations in astrocytes in post-status animals it is believed that this switch in spatial scale of Ca+2 signaling results from ambient activation of mGluR5s, presumably due to an enhanced level of external glutamate that accumulates during the process of cell death that follows status epilepticus. This can be confirmed in cortical and hippocampal astrocytes using two-photon microscopy and photolysis to study Ca+2 signaling in individual astrocytes.
[0157] Using the recently developed ability to image Ca+2 levels in hippocampal astrocytes in vivo the extent to which this stimulus for gliotransmission is modified after status epilepticus can be determined. Pilocarpine-induced status epilepticus can be induced in mice, and hippocampal astrocytic Ca+2 imaged 2-7 days later to determine the degree to which excitability is modified. The mice are to be maintained until they become epileptic, whereupon astrocytic Ca is imaged, hi previous studies imaging astrocytes in the cortex, it was found that cortical astrocytic Ca+2 signaling was similar to that of control animals when animals had become epileptic. Since it is unlikely that the source of these seizures is in the region of the cortex that was imaged, it is not surprising that there was no change in the astrocytic Ca+2 signal. However, the hippocampus can be the focus of epileptiform activity in epileptic animals. Therefore, in addition to measuring glial Ca+2 signaling during the latent period of
. lu ^ . sit :» c: .:;: . , „ , , ,. - , , epileptogenesis, video-monitoπng of mice can also be performed to determine when they become epileptic. After identifying mice as epileptic, 24h monitoring can be performed to select mice that have not had a seizure during the previous day (to prevent acute effects of a seizure) for the imaging of astrocytic Ca+2. With these animals it can be determined whether astrocytic Ca+2 signaling, and thus gliotransmission, are enhanced in the hippocampus of epileptic mice.
[0158] After identifying changes in Ca+2 signaling in hippocampal astrocytes follow-up experiments can be performed as previously described for cortical astrocytes. The relevant questions are whether the mGluR5 antagonist MPEP reduces these Ca+2 oscillations, and whether DHPG, an mGluR.5 agonist, induces cell-wide Ca+2 oscillations.
[0159] These experiments are expected to identify changes in astrocytic Ca+2 signal in hippocampus similar to those already detected in cortical astrocytes.
Example 8
Determination of Whether Global Activation of mGluR5 Receptors Switch Local Ca+2 Signals to Cell- Wide Events in Astrocytes from Control Animals
[0160] The preliminary results that suggest that mGluR5 activation results in cell- wide Ca+2 oscillations can be extended as follows. Ca+2 signaling in bulk-loaded fluo-4-containing astrocytes in vivo can be imaged. Fluo-4 intensity can be imaged to indicate Ca+2 signals and, after obtaining a 300-second control period of images from up to eight astrocytes per animal, DHPG (2OuM) can be added to the cortical surface, followed by relocation of each astrocyte using vasculature landmarks, after which a second period of imaging can be performed. Ca+2 signals can be measured within processes as well as the cell body to determine the degree to which Ca+2 signals are coordinately regulated within a cell by performing a cross correlation analysis of the Ca+2 signals within distinct process of the cell before and after addition of DHPG. [0161] The goal of adding DHPG is not to fully activate mGluR5, but instead to provide a partial activation of these receptors on which subsequent inputs can summate. Therefore, a dose response relationship should be performed to determine the minimal concentration of DHPG required to switch local to global Ca+2 signals. To determine which receptors are mediating the actions of DHPG, the mGluR5 selective antagonist MPEP can also be applied, which will determine whether the observed effects result from mGluR5 activation.
[0162] These experiments are expected to show that addition of DHPG will lead to a change in local to cell-wide Ca+2 signals as suggested by preliminary studies
[0163] Because little spontaneous synaptic activity has been observed where the above- mentioned experiments are carried out in brain slices, thereby reducing the excitatory drive to the
astrocyte, an alternative strategy is to provide focal stimulation, through a small extracellular pipette, to a small group of Schaffer collateral afferents adjacent to the processes of an astrocyte. In this manner local Ca+2 signals can be evoked wherein it can be investigated whether increasing concentrations of DHPG increase the range over which these astrocytic Ca+2 signals spread.
Example 9
Determination of Whether Basal Activation of mGIuR5 Switch Local Ca+2 Signals, Induced by Photo-Release of Caged MNI Glutamate, into Cell- Wide Oscillations
[0164] In this example, photolysis is used to stimulate a local region of cellular processes and determine the extent to which the Ca+2 signal spreads through the arbor of the astrocyte. Given the evidence of the capacity to photo-release caged compounds in vivo, the goal is to perform this experiment entirely in vivo.
[0165] Caged MNI-glutamate (ImM) can be introduced into the cortex in vivo, either through application to the cortical surface or through a focal ejection from a pipette, and can focally photo-release glutamate onto a region of the astrocytic processes to evoke a local Ca+2 signal. The extent of spread of the Ca+2 signal can then be determined to confirm that it is due to activation of mGluR5 by co-application of MPEP (tail vein injection). In separate experiments, in which MPEP has not been applied, glutamate can be photoreleased and, after determining the extent of spread of the Ca+2 signal, the cortical surface DHPG can be applied at increasing concentrations from 1 to 50μM, although the exact concentration may vary depending on the results of the experiments detailed in Example 7.
[0166] It should be determined whether DHPG increases the range of spread of the photolysis-evoked Ca+2 response. Additionally, a glutamate dose-response curve should be performed in which the magnitude of the local Ca+2 signal in relation to the photolysis power is determined, and to determine whether concentrations of DHPG that alter the spread of the Ca+ signal also reduce the power required to evoke a Ca+2 signal. Such studies should be performed at constant depth in the cortex to set forth a constant amount of scattering of excitation energy between different experiments. It is anticipated that low concentrations of DHPG will enlarge the distance over which focally-released glutamate will evoke a Ca+2 signal.
[0167] One potential obstacle to such experiments is the ability to perform photolysis in vivo. Thus, the same experiment may alternatively be carried out using brain slices where success with photolytic application of glutamate and the activation of glial Ca+2 signals has been previously demonstrated.
[0168] An additional experiment in which caged IP3 is introduced into a single astrocyte together with fluo-4 by patch pipette delivery may also be performed. In this experiment IP3 will be focally released within a region of the cellular to determine whether DHPG application alters the extent of spread of the photolysis-induced Ca+2 signal. The advantage of this experiment is that a Ca+2 signal can be produced independent of mGluR5 activation, thereby allowing independent control of receptor and IP3 levels. A disadvantage of the approach may be the experiments performed in brain slices to allow reliable introduction of caged compound by a patch pipette into an astrocyte. As an alternative to caged IP3 and caged MNI-glutamate, caged serotonin can be used. With caged serotonin, studies will be able to be performed in vivo having independent control over the receptor activated by photolysis and by DHPG application.
Example 10 Determination of Whether the mGIuR5 Antagonist, MPEP, Reduces Cell- Wide Ca+2
Signals Detected in Post-Status Animals into Local Ca+2 Signals [0169] Animals were used three days following status epilepticus when cell-wide Ca+2 oscillation frequency is elevated. As described above, Ca+2 signals were monitored within astrocytic processes and the cell body for 300s. Up to eight astrocytes per animal were studied. MPEP was injected into the tail vein and, after waiting 20 min for access to the nervous system, the identified astrocytes was re-imaged to determine whether Ca+2 oscillation frequency, as assessed by the cell body signal, was reduced and whether MPEP switches the cell-wide Ca+2 oscillations to local signals within their processes.
Example 11
Determination of Whether the mGluR5 Antagonist, MPEP, Reduces the Spatial Extent to which Photolysis of Caged IPτ-Induced Ca+2 Signal will Spread in an Astrocyte
[0170] To determine whether the activation of mGluR5 is responsible for causing the cell-wide Ca+2 signals seen in astrocytes in post-status animals, caged IP3 (delivered from a patch pipette) will be focally released into the interior of an astrocyte. It can then be determined whether subsequent addition of MPEP reduces the extent to which photolysis evokes a Ca+2 signal. If there is an elevated basal extracellular glutamate level, then it is predicted that this will lead to an overall increase in resting IP3 level in the astrocyte. Though this small change may be sub-threshold for inducing significant Ca+2 signals, when it is additive with a pulse of IP3 it is
-11IL... i / a / ιι '..:;: .(;; predicted that Ca may be released over a larger region of the cell. As an alternative to caged
IP3, caged serotonin is available, which can be used in vivo.
[0171] Because of potential movement artifacts changing the fluorescent intensity recorded from single wavelength Ca+2 indicators, the ratio of the intensity of fluo-4 against the intensity of the Ca+2-insensitive dye sulforhodamine 101 should be determined. Because slight movement will result in equivalent changes in intensity of both dyes, ratio measurements will prevent errant signals being generated. In preliminary studies it has been determined that this is a feasible approach and that Ca+2 signals can be detected (data not shown). .
[0172] To control for effects of injection of pilocarpine, effects in animals that have been injected with pilocarpine to induce status epilepticus can be compared with effects observed in animals injected with l/10th the concentration of pilocarpine, which does not result in status epilepticus, or with animals injected with vehicle alone. In the photolysis studies, potential photodamage can be controlled for by repeating experiments with the same laser intensity but in the absence of caged compound. Additionally, as discussed above, it will also be an important control to determine whether the mGluR5 antagonist MPEP blocks Ca+2 signals induced by the photolysis of caged MNI-glutamate.
Example 12
Determination of Whether Activation of mGluR5 Evokes Ca+2 Oscillations in Astrocytes and Ifenprodil-Sensitive NMDA Currents in Area CA3 and Layer 2/3 Pyramidal Neurons
[0173] Brain slice studies have demonstrated that area CAl hippocampal pyramidal neurons receive direct excitation from astrocytes; Ca+2 oscillations in these glial cells evoke the release of glutamate that acts through ifenprodil-sensitive NR2B subunit-containing NMDA receptors to cause SICs that can, when recorded in current clamp mode, lead to synchronous depolarizations of groups of pyramidal neurons. Since the NR2B -containing NMDA receptors have been linked to cell death pathways, this glial-induced neuronal excitation has the potential, if over-utilized, to induce pathological neuronal responses. Because neuronal death is detected in layer 2/3 cortical neurons and in area C A3 of the hippocampus, it is essential to determine whether astrocyte-evoked SICs are present in these locations in addition to area CAl and to further determine whether the NR2B-containing NMDA receptor mediates these currents.
[0174] Previous work had demonstrated that area CAl neurons are directly excited by glial glutamate acting through NR2B-containing NMDA receptors (Fellin et al. 2004). It is desired to extend this initial observation to areas C A3 of the hippocampus and to area 2/3 of the cortex, two regions where delayed neuronal death has been observed following status epilepticus.
Because the experimental apprόacn iόf'area CA3 and. layer 2/3 of the cortex are identical, the description below does not distinguish among either region unless there are region-specific details that need to be defined.
[0175] Brain slices were prepared from control animals and paired whole-cell recordings were made from pyramidal neurons to monitor for astrocyte-evoked SICs while simultaneously imaging astrocytic Ca+2. While recording from neurons the characteristic time- course of SICs, which exhibit a slower rise time than synaptic currents (~70ms compared to ~2ms) as well as a slower decay time (450ms compared to <10ms) were monitored. Under normal physiological conditions, astrocyte-evoked SICs were detected only in about 30% of the neurons and at a low frequency of about one event every 10 min. Without intending to be limited to any particular theory or mechanism of action, it is believed that this paucity of events arises from the fact that local microdomain Ca+2 elevations in astrocytes, rather than cell-wide oscillations, are the norm in physiological tissue. Because microdomain Ca+2 elevations will recruit few release sites they will evoke small, barely-detectable currents in normal physiology. Nevertheless, the low frequency of cell-wide astrocytic Ca+2 elevations observed in control tissue was still sufficient to evoke detectable SICs and permitted the determination of whether astrocytes evoke these neuronal currents in area CA3 and layer 2/3, as they do in area CAl.
[0176] To increase the probability of detecting astrocyte-evoked neuronal SICs, the mGluR was superperfused agonist DHPG (lOuM), which in area CAl increases cell-wide astrocytic Ca+2 oscillation frequency, to determine whether cell-wide astrocytic Ca+2 oscillation frequency and SIC frequency were augmented. Additionally, it was determined whether the actions of DHPG are mediated by mGluR5 by co-applying, in some experiments, the mGluR5 antagonist MPEP (5OuM).
[0177] In addition to using DHPG as a stimulus for astrocytes, other stimuli that are known to elevate cell-wide astrocytic Ca+2 oscillation frequency are available. For example, removing added Ca+2 to ACSF increased astrocytic Ca+2 oscillation frequency, although the mechanism behind this phenomenon is not clear. Because this manipulation also reduces synaptic transmission, it has been a useful stimulus to activate astrocytic pathways, hi area CAl, zero added Ca+2 ACSF stimulated astrocytic Ca+2 oscillations and increased the frequency of SICs. Additionally, astrocytic Ca+2 signals were activated by the addition of ATP (lOuM), while synaptic transmission was depressed. Thus, this serves as an alternative stimulus to activate astrocytic Ca+2 signals and evoke SICS.
[0178] Because kinetics alone were not sufficient to determine whether astrocytes evoke neuronal NMDA currents, a pharmacological study was performed to determine whether
area CAl . Previously, it had been shown that astrocyte-evoked neuronal SICs are insensitive to both tetrodotoxin and the AMPA antagonist CNQX, and insensitive to acute treatment with the clostridial toxin tetanus toxin (Fellin et ah, 2004). In contrast, SICs were blocked by the NMDA receptor antagonist D-AP5, augmented in frequency and magnitude by removal of external Mg+2, and sensitive to the NR2B subunit containing NMDA receptor antagonist ifenprodil (Fellin et al, 2004). Table 2 shows the distinctive pharmacological characteristics that, together with their distinctive kinetics, allow the detection of SICs as arising from an astrocyte source. These pharmacological tools confirmed the NR2B subunit containing nature of the NMDA response of SICs.
Table 2. Pharmacological and kinetic fingerprint of astrocyte-evoked SICs in comparison to syna tic currents.
[0179] Initial studies were performed using slices less than three weeks of age because this age both facilitates whole-cell recordings and because Ca+2 indicators are readily bulk- loaded into astrocytes at these or younger ages. However, later experiments used slices from adult animals, which provided lower yield results but confirmed studies performed at the younger ages.
[0180] Studies demonstrated slow kinetic, SIC-like events both in area CA3 and in layer 2/3 cortical neurons. Moreover, during the studies, it was determined that these SIC-like events are insensitive to CNQX and TTX and sensitive to D-AP5. Though the n values are low, it seems from these studies that astrocyte-evoked neuronal SICs are a common property in several regions of the nervous system. The above-mentioned experiments revealed an overall similar pharmacological fingerprint as defined for area CAl pyramidal neurons.
[0181] Because it has been determined that the addition of DHPG permits cell-wide astrocytic Ca+2 oscillations in layer 2/3 of the cortex in vivo, it is anticipated that low concentrations of DHPG will allow microdomam Ca+2 elevations to become cell-wide events and will thus increase the frequency of observing large amplitude astrocyte-evoked neuronal SICs.
Example 13
Determination of Whether Photolytic Elevation of Astrocytic Ca+2 Evokes Ifenprodil- Sensitive NMDA Currents in Area CA3 and Layer 2/3 Cortical Pyramidal Neurons
[0182] In this Example, photolysis is used to increase astrocytic Ca+2 and to determine whether neuronal detected NMDA currents are observed. This approach has been previously carried out for area CAl (see Fellin et ah, 2004). Cortical and hippocampal slices can be co- loaded with the Ca+2 indicator fluo-4 and the calcium cage NP-EGTA by incubation in their membrane-permeant acetoxymethyl esters. Previous studies have demonstrated that these compounds selectively load into astrocytes in slices. Subsequently, a whole-cell recording from a pyramidal neuron will be performed using a patch pipette filled with Alexa 568 (5OuM) to disclose the locations of its dendrites.
[0183] A 3μm spot of UV energy can be illuminated onto a cell body of an adjacent astrocyte, and the relative Ca+2 level can be monitored using fluo-4 thereby permitting the determination of when the Ca+2 signal propagates to the region of the dendrite. Simultaneous electrophysiology can be used to identify the presence of astrocyte-evoked currents. Since previous studies have shown that SICs can be repetitively evoked, prior to subsequent photolysis pulse, antagonists as ifenprodil and D-AP5 can be included in the superfusate to determine whether CA3 and cortical layer 2/3 astrocytes evoke NR2B subunit-containing NMDA responses in adjacent pyramidal neurons.
Example 14
Determination of Whether Blockade of Ca+2 Signaling and Glutamate Release from Astrocytes Prevents Ifenprodil-Sensitive Neuronal SICs
[0184] An additional experiment can be performed in which molecular genetics is used to provide unequivocal evidence that cell-wide Ca+2 oscillations in astrocytes evoke neuronal SICs. Two molecular genetic manipulations can be used in this regard — an existing astrocyte- specific transgenic animal which impairs glutamate release from astrocytes (the astrocyte- specific SNARE animal), and viral expression of RGS2 specifically in astrocytes to block their receptor-activated Ca+2 signals. These experiments will provide a determination of whether these astrocyte-specific manipulations impair neuronally detected astrocyte-evoked NMDA receptor-dependent SICs .
[0185] Brain slices obtained from transgenic animals, wild-type littermates, or from wild-type animals previously injected with adenovirus to induce the expression of RGS2-EGFP
under the control of a GFAP promoter, can be used to simultaneously study the ability of the mGluR5 agonist to induce cell-wide astrocytic Ca+2 signals as well as neuronally detected NMDA receptor-dependent SICs. Because preliminary studies have already shown significantly reduced glutamate release in cultures from the astrocyte-specific SNARE animal, it is anticipated that the resting and DHPG-induced frequency and amplitude of SICs will both be reduced when compared to wild-type littermates and transgenics maintained on doxycycline to suppress the expression of the dominant negative SNARE domain. In contrast, it is expected that the expression of the transgene will have no impact on the astrocytic Ca+2 signal as this is upstream of the glutamate release machinery.
[0186] When RGS2-EGFP is virally expressed in astrocytes, it is expected to block, or attenuate, the ability of DHPG to evoke a Ca+2 response in astrocytes, and will similarly prevent astrocyte-dependent Ca+2-evoked neuronally detected SICs. hi this experiment the Ca+2 signal and SIC parameters can be compared to those recorded in adjacent slices from the same animal that were not infected by the virus and additionally compared to control animals that received viral injections in which GFAP promoter regulates EGFP expression only.
[0187] One potential concern with the above-mentioned experiments is that the SNARE domain suppresses the release of ATP as well as glutamate from astrocytes, hi previous studies using this animal, it was shown that the predominant effect of the reduced ATP release manifests ■ itself as an increase in the amplitude of excitatory synaptic transmission; released ATP is normally degraded by ectonucleotidases to adenosine which causes a persistent Al receptor- mediated suppression of synaptic transmission. These studies have also shown that the effects of transgene expression on ATP and adenosine can be fully reversed by exogenous addition of the Al receptor agonist, CCPA (1OnM). To counter this problem, two control experiments can be performed. First, the Al receptor antagonist DPCPX (80OnM) can be included in experiments using wild-type and transgene-expressing slices so that, regardless of whether the transgene is expressed, the persistent Al activation is absent. Second, the Al agonist, CCPA (10-5OnM), can be included in the ACSF to recover the persistent Al activation in transgene-expressing slices.
[0188] A second potential concern is that transgene expression is weak in the cortex relative to the hippocampus. Therefore, in experiments focused on the cortex it is important to determine whether sufficient transgene is expressed in astrocytes to block glutamate release from astrocytes. If no effect on glial glutamate release is observed, the viral expression of RGS2 alone can be used in cortical experiments. Indeed, because multiple strategies to manipulate glial glutamate release, there is some redundancy built into the experimental design to overcome potential problems. If RGS2 is determined to be expressed at levels insufficient to inhibit Gq-
mediated, astrocytic Ca signaling, a backup with mutant Gqα subunits is available. In one of these, the Gqα is in a persistently GTP-bound form. Because of the persistent activation, it is believed that internal Ca+2 stores are depleted leading to a blockade of receptor-dependent astrocytic Ca+2 signaling.
[0189] A third potential concern is how widespread transgene expression will be following adenovirus injection and whether there might be cytotoxic effects from the adenovirus. Though success has been obtained with the adenovirus technique, astrocyte-specific lentiviral vectors are currently in development that can serve as alternatives. In fact, Tetracycline- regulated lentivirus constructs that permit the use of doxycycline to control the expression of the genetically encoded Ca+2 indicator, VC6.1, have already been developed to study transplantation of stem cells into the cortex.
[0190] Thus, an alternative strategy is to develop a tetracycline-regulated RGS lentiviral construct. Rather than cloning the GFAP promoter into this construct the existing tetO promoter can be used, and, after introducing RGS-EGFP, the virus can be injected into the hippocampus and cortex of transgenic animals in which the GFAP promoter drives the expression of the transactivator, tTA, selectively in astrocytes. This can provide cell-type specific, doxycycline regulation of the transgene RGS2. In this manner there can be exquisite control over transgene expression by addition of doxycycline to the animals' drinking water, which will allow the control of potential cytotoxic effects of viral injection.
Example 15 Determination of Whether the Astrocyte-Evoked Neuronal Excitation Pathway is Intact
Following Status Epilepticus
[0191] Because membrane receptors can be internalized during status epilepticus, it is important to determine whether each of the signaling elements in the gliotransmission pathway is intact following status epilepticus. Since it has been determined that the astrocytic Ca+2 signaling pathway is functional, whether astrocytic Ca+2 signals stimulate glutamate release detected by neuronal NR2B-containing NMDA receptors was evaluated.
[0192] Brain slices were isolated from animals 1-3 days following status epilepticus and perform experiment similar to those Examples above. Spontaneous and DHPG-evoked astrocytic Ca+2 signals and the presence of SICs were recorded. As a follow-up, whether SICs are D-AP5 and ifenprodil sensitive was detennined, along with whether the expression of the dominant negative SNARE domain in astrocyte blocks this pathway, and whether the expression of RGS2 blocks astrocytic Ca+2 signaling and SICs. By performing this experiment on tissue
isolated from animals 1-3 days following status epilepticus, the eπectiveness of the reagents that used to perturb this pathway in vivo was determined in addition to demonstrating the presence of gliotransmission. λ
Example 16
Determination of the frequency of Unstimulated Neuronal SICs Elevated in Tissue Isolated from Animals 1-3 Days Following Status Epilepticus
[0193] It is predicted that the frequency of astrocytic Ca+2 oscillations and SICs would be increased for up to three days following status epilepticus. This can be tested on brain slice preparations from animals 1-3 days after pilocarpine injection and status epilepticus, and the frequency of astrocytic Ca+2 oscillations measured in the cell body and the frequency of neuronally-detected SICs can be determined.
[0194] SICs can be identified on the basis of their kinetics. Sensitivity to D-AP5 and ifenprodil and insensitivity to TTX and CNQX can be used to identify SICS. Measurements are made in ACSF (rest) during application of DHPG, and during application of the niGluR5 antagonist MPEP. It is predicted that there will be an elevated frequency of Ca+2 oscillations under resting conditions due to cell-wide Ca+2 oscillations, and that DHPG will have little further stimulatory effect on either astrocytic Ca+2 or SIC frequency. It is further predicted that MPEP will reduce the frequency of both the Ca+2 oscillation and the SIC.
Example 17
Determination of Whether Pharmacological Manipulation of the Gliotransmission Pathway
Will Reduce Delayed Neuronal Excitotoxicity
[0195] Because an enhanced period of astrocytic Ca+2 excitability is temporally coincident with the delayed neuronal death that follows status epilepticus, it is believed that the these Ca+2 oscillations, acting through glial glutamate release, evoke NMDA receptor-dependent neuronal excitotoxicity. Pharmacological and molecular genetic manipulations that suppress this gliotransmission pathway can be used to test the causal relationship between gliotransmission and delayed neuronal death.
[0196] Preliminary studies have provided evidence to support the notion that intraperitoneal injection of MPEP or ifenprodil, both of which impact the gliotransmission pathway, leads to a reduction in the delayed neuronal death that follows status epilepticus. Such studies can be extended to determine the dose-response relationships as well as time course of this suppression of excitotoxicity.
,., i : ::: .,::
[0197] In this Example, Pilocarpine is injected into mice to evoke status epilepticus.
After a duration of one hour, the seizure is terminated by injection of diazepam. After a further one hour period the test pharmacological agent can be injected into the animal, and the animal returned to its cage. The antagonist can be re-injected into the animal at successive daily intervals, for a total of four injections. It has previously been determined that this injection protocol significantly reduces neuronal death. Animals are to be sacrificed at 1, 3, 5, 7 and 10 days following status epilepticus (5 animals in each group). The animals can then be cardiac- perfused with 4% paraformaldehyde, and sections of cortex and hippocampus (20um) can then be cut and reacted with Fluoro-Jade B (Schmued and Hopkins, 2000) to disclose dying cells. In a parallel study, two additional groups of animals are to be examined, hi the first, a subthreshold dose of pilocarpine is injected into the animal, and the number of dying cells at each time point after injection can then be examined, hi the second, pilocarpine is injected to evoke status epilepticus, and subsequently, vehicle injections can be made in place of pharmacological antagonists.
[0198] For these experiments, a series of one in five sections of the hippocampus and cortex for Fluoro-Jade B, hematoxolyin, anti-GFAP, anti-NeuN and ApopTag reactions can be collected. These sections can then be used to examine the extent to which death is induced (fluorjade-B and ApopTag) and to determine when astrocytes become reactive (GFAP). In two Fluoro-Jade B sections from each animal triple label reactions can be performed to determine whether the dying cells are neurons (anti-NeuN) or astrocytes (GFAP). Delayed death can be counted in the entire areas CA3, CAl and layer 2/3 of the cortex in each of four separate sections. Based on Fluoro-Jade B labeling the density of Fluoro-Jade B labeling per μm2 can be estimated. Results at each time point and condition can then be analysed using an ANOVA test followed by t-test. The dose-response relationship can be measured by reducing the concentration of injected antagonist at 0.5 log unit increments in the range defined by Table 3.
Table 3. Pharmacological agents, routes of administration, and concentrations that can be used.
[0199] There are predictable outcomes on astrocytic Ca signaling of the pharmacological treatments that will be important to use as controls. First, animals (four in each group) can be evaluated to confirm that each tested concentration of MPEP reduces the astrocytic Ca+2 oscillation frequency and amplitude. It is expected that MPEP will prevent the astrocytic Ca+2 oscillations as well as reduce neuronal death. Indeed, in studies in which MPEP (lmg/kg) was administered for three days following status epilepticus it was found that astrocytic Ca+2 oscillations were significantly reduced compared to animals that had status epilepticus but that received only a vehicle treatment following this seizure.
[0200] An additional experiment that can be performed is to determine whether ifenprodil prevents the increase in astrocytic Ca+2 oscillations that normally follow status epilepticus. Since ifenprodil acts downstream of gliotransmission on the target of glial glutamate action, the NR2B-containing NMDA receptor, acute administration of ifenprodil does not reduce astrocytic Ca+2 oscillations when administered at the time of imaging astrocytic Ca+2 three days following status epilepticus. However, it should be determined whether daily treatment with ifenprodil, which preliminary experiments suggest significantly suppresses delayed neuronal death, prevents the increase in astrocytic Ca+2 oscillations that are normally seen following status epilepticus. Since astrocytes do not express NMDA receptors, this result would suggest that there is a positive feedback loop in which initial injury stimulates astrocytic Ca+2 oscillations which in turn provide further glutamate to act on the ifenprodil-sensitive NMDA receptors.
Example 18
Determination of Whether Astrocyte-Specific Molecular Manipulation of the Gliotransmission Will Reduce Delayed Neuronal Excitotoxicity
[0201] It is expected that the experiments in Example 25 will provide significant new insights into the role of mGluRs and NR2B-containing NMDA receptors in mediating delayed neuronal death. While these studies should implicate the astrocyte as a contributor to neuronal death, unequivocal identification of such a role will require astrocyte-specific manipulations to prevent the activation of gliotransmission. Thus, astrocyte-specific transgenic animals and virally expressed RGS2 can be used to block astrocyte-glutamate release and Gq-evoked Ca+2 signaling respectively, in order to confirm that astrocytes contribute to delayed neuronal death.
[0202] hi this Example, transgenes will be expressed in astrocytes to block glutamate release and Gq-dependent Ca+2 signaling, instead of injecting antagonists after status epilepticus and determining consequences for delayed neuronal death. Because it may not possible to instantaneously turn-on or off the expression of the transgene. Thus it may be necessary to
;:;ii. ,ι i . , . , . ., . control the expression of the transgene prior to the induction of status epilepticus then determine consequences of astrocyte-specific transgene expression on delayed neuronal death. The exact conditions for the best expression of these transgenes is discussed in Example 22, above.
[0203] As set forth in Example 25, astrocytic Ca+2 signals can be measured following status epilepticus to determine whether RGS2 expression suppresses cell-wide Ca+2 oscillations and to determine whether the inhibition of gliotransmission by SNARE domain expression in astrocytes prevents the augmentation of Ca+2 signaling. If SNARE domain expression blocks or reduces the increase in Ca+2 signaling together with reducing delayed neuronal death, this would be consistent with a positive feedback control of glial Ca+2 signaling as discussed in Example 25 when ifenprodil is administered to animals. Briefly, if astrocyte-specific SNARE domain expression retards neuronal death and reduces the increase in Ca+2 signaling normally detected in animals following status epilepticus this would suggest that an initial neuronal damage event increases astrocytic Ca+2 signals which in turn feed back to stimulate further neuronal damage. Anti-GFAP immunoreactivity can be used to confirm that transgenes are selectively expressed in astrocytes.
[0204] Given the ability of MPEP and ifenprodil to reduce delayed neuronal death following status epilepticus it is expected that transgene expression in astrocytes will similarly reduce this outcome measure. One complicating issue in data interpretation is whether astrocyte-specific inhibition of gliotransmission affects the magnitude of status epilepticus, and therefore whether consequences of transgene expression on delayed neuronal death result from actions on status epilepticus or on delayed death per se. Thus, the severity of status epilepticus must be compared, and it must be determined whether the dose of pilocarpine musts be changed to obtain a similar magnitude action. This will be especially important when using transgenic animals where the majority of astrocytes express this transgene. However, in virally expressed RGS2 animals, it will be less of a problem because Gq-mediated signaling should be inhibited in only in a subset of astrocytes. Thus, it is expected that pilocarpine should cause the same magnitude of status epilepticus as in control animals, and conclusions can ultimately be based on a combination of pharmacological treatments together with astrocyte-specific transgenic animals and virally expressed RGS2 studies.
[0205] One potential problem with the use of the astrocyte-specific dominant negative SNARE animals is that the expression of this transgene reduces ATP release as well as the release of glutamate. Since previous studies have shown that ATP is rapidly degraded to adenosine, which causes a persistent suppression of excitatory transmission, it will be important to control this pathway in transgenic and wild-type littermates. Fortunately, the Al receptor
- u a ι •» antagonist DPCPX is known to cross me blood brain barrier when administered systemically.
Therefore in wild-type and transgenic animals, maintained with or without doxycycline, DPCPX (lmg/kg) or the Al agonist CCPA (lmg/kg) can be administered systemically to the animal on a daily basis by intraperitoneal injection. Since the transgene, and thus DPCPX administration, can be expressed at the time of inducing status epilepticus, the concentration of pilocarpine can be adjusted accordingly to achieve the same degree of status epilepticus in these studies. As an alternative strategy, osmotic mini-pumps can be used for administration of pharmacological agents into the nervous system. Pumps can be used for sustained administration of Al pharmaceuticals, as well as for glutamatergic pharmaceuticals, as appropriate, since this approach should prevent unwanted systemic effects of pharmaceuticals.
Example 19
Determination of Whether Pharmacological Manipulation of the Gliotransmission Pathway
Will Suppress Epileptogenesis
[0206] It is known that, following status epilepticus induced by pilocarpine administration, a latent period ensues during which undefined cellular and molecular reorganizations lead to the animal developing epilepsy. The next step is to determine whether gliotransmission induced by the enhanced Ca+2 excitability of the astrocyte is responsible in part for epileptogenesis by the use of pharmacological and molecular genetic manipulations to perturb the gliotransmission pathway.
[0207] In this Example, Pilocarpine is injected into mice to evoke status epilepticus, and after one hour, the seizure is terminated by injection of diazepam. After a further one hour period the test pharmacological agent (e.g., MPEP and ifenprodil) is injected into the animal, and the animal is returned to its cage. At daily intervals, the antagonist can be re-injected for between three and seven days. Animals can be maintained for 30-90 days, and the degree to which the animals develop spontaneous seizures can be assessed.
Example 20 Inhibition of SNARE Expression Facilitates Adenosine Release and Produces
Anticonvulsive Effects
[0208] Transgenic mice that selectively express a dnSNARE transgene under control of a tetracycline regulator only in astrocytes were used to study the release of astrocyte-dependent accumulation of adenosine in epileptic animals. Animals were maintained on the tetracycline analog Doxycycline to prevent dnSNARE expression (Figure 18). Animals were maintained in
this manner, then status epilepticus was induced and animals maintained until at least one month later when they became epileptic. At this point animals were videotaped to monitor seizure frequency. Data are shown for three sequential analysis periods, performed within the same animals.
[0209] Initially seizure frequency was monitored while on a diet containing doxycycline to prevent transgene expression. Doxycycline was then removed, to allow dnSNARE expression, seizure frequency again assessed, then doxycycline was added back to the diet to prevent dnSNARE expression and again seizure frequency was assessed. The seizure frequency was found to increase when astrocytes did not regulate adenosine accumulation (doxycycline removed from diet and dnSNARE expressed in astrocytes). This result clearly demonstrates that the regulation of accumulation of the natural anticonvulsant adenosine from astrocytes controls epileptic seizures. It should be noted that in this experiment treatments began after epilepsy had commenced and that selective manipulation of astrocytes and their control of adenosine accumulation can be used to control seizures.
Example 21 Chelation of Cellular Calcium in Astrocytes Attenuates Neuronal Death
[0210] Status epilepticus was induced in mice using pilocarpine. Three hours following status epilepticus, mice were anesthetized using Xylazine/ketamine. A cranionatomy was performed on one side of barrel cortex and the meninges was carefully removed. The contralateral cortical region in the same mouse was used as control. Drug was applied 4-5 hours after entering status epilepticus. Exposed cortical regions was exposed to HEPES buffed ACSF containing BAPTA-AM( 20OuM) or U 7 compound for 5minutes. Then 50ul of 2% agarose containing the same drug was put into the exposed cortical region. After the agarose solidified, the skin was sealed. Mice were allowed to recover in the heating pad before returning to the cage. 3 days later, mice were transcardially perfused. Cell death was assessed as described before using fluorojade B (FJB)..
[0211] Figure 19 shows that treatment of the cortex with BAPTA/AM, which selectively chelates Ca+2 in astrocytes, attenuates neuronal death as assayed by FJB. hi comparison to contralateral cortex, which did not receive BAPTA/AM treatment, neuronal death was significantly attenuated by BAPTA/AM treatment. Because BAPTA/AM selectively loads into astrocytes to chelate their internal Ca+2 and prevent Ca+2 oscillations and gliotransmission this data provides a direct demonstration that astrocytes are responsible for the delayed neuronal death that follows status epilepticus. P<0.05
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[0212] The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.
Claims
What is Claimed:
1. A method for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of at least one protein that participates in calcium signaling within astrocytes.
2. The method of claim 1 , wherein the protein is mGluR receptors, Gq, phospholipase C, Regulator of G Protein Signaling, IP3 phosphatase, IP3 Receptor, P2Y1, P2Y2, P2Y4, PIP2 kinase, PIP2 phosphatase, or calbindin.
3. The method of claim 1, wherein the chemical transmitter is glutamate, adenosine, ATP, or D-serine.
4. The method of claim 1, wherein the condition is epileptogenesis, epilepsy, convulsions, schizophrenia, excitotoxic damage, demyelination, brain ischemia, neuronal death, motor impairment, attention deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HTV dementia, bipolar disorder, FTDP- 17, Hepatic encephalopathy, or Lupus encephalitis.
5. The method of claim 1, wherein the modulation comprises inhibiting at least one gene encoding the proteins.
6. The method of claim 5, wherein the inhibiting comprises administering to the subject an antisense oligonucleotide, RNAi, or siRNA.
7. The method of claim 1, wherein the modulation comprises administering to the subject a therapeutically effective amount of at least one antagonist to at least one of the proteins.
8. The method of claim 7, wherein the antagonist is MPEP, MTEP, SIB-1893, SIB-1757, GPant-2A, PPADS, or U-73122.
9. The method of claim 2, wherein the mGluR receptor is mGluR5.
10. The method of claim 2, wherein the Regulator of G protein signaling is RGS2.
11. The method of claim 1 , wherein the subj ect has suffered a traumatic injury to the nervous system.
12. A method for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of at least one protein that participates in the expression or release of glutamate.
13. The method of claim 12, wherein the protein is SNARE proteins, synaptotagmin IV, Munc-18, VGLUTl, VGLUT2, or VGLUT3.
14. The method of claim 12, wherein the condition is epileptogenesis, epilepsy, ADHD, depression, or schizophrenia.
r . ,. ,™ -.„
15. Trie method of claim 12, wherein the modulation compnses inhibiting at least one gene encoding the proteins.
16. The method of claim 15, wherein the inhibiting comprises administering to the subject an antisense oligonucleotide, RNAi, or siRNA.
17. The method of claim 12, wherein the modulation comprises administering to the subject a therapeutically effective amount of a Clostridial toxin.
18. The method of claim 17, wherein the Clostridial toxin is tetanus toxin, botulinum toxin A, botulinum toxin B, botulinum toxin Cl, botulinum toxin D, botulinum toxin E, botulinum toxin F, or botulinum toxin G.
19. The method of claim 12, wherein the modulation comprises administering to the subject a therapeutically effective amount of at least one antagonist to at least one of the proteins.
20. The method of claim 19, wherein the antagonist inhibits formation of a SNARE complex.
21. The method of claim 12, wherein the subj ect has suffered a traumatic injury to the nervous system.
22. A method for treating or preventing a neurological or psychiatric condition in a subject comprising inhibiting the expression or activity of at least one protein that is activated by glutamate.
23. The method of claim 22, wherein the protein is synaptic.
24. The method of claim 22, wherein the protein is extrasynaptic.
25. The method of claim 22, wherein the protein is NMDA receptors or AMPA receptors.
26. The method of claim 22, wherein the inhibiting comprises administering to the subject a therapeutically effective amount of at least one antagonist to at least one of the proteins.
27. The method of claim 26, wherein the antagonist is D-AP5, (R)-AP5, PBPD, EAB515, LY233536, MK-801, Memantine, Ketamine, L-701,324, L-689,560, GV196771A, Ro 25-6981, Co 101949, or Ifenprodil.
28. The method of claim 26, wherein the antagonist interacts with the NR2B subunit of the NMDA receptor.
29. The method of claim 22, wherein the condition is epileptogenesis, epilepsy, neuronal death, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP- 17, Hepatic encephalopathy, or Lupus encephalitis.
30. The method of claim 22, wherein the subject has suffered a traumatic injury to the nervous system.
31. A method for treating or preventing a neurological or psychiatric condition in a subj ect comprising modulating the oscillation of free cellular calcium in astrocytes.
32. The method of claim 31 , wherein the condition is epileptogenesis, epilepsy, schizophrenia, excitotoxic damage, demyelination, brain ischemia, neuronal death, motor impairment, ADHD, Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, cerebrovascular disease, hydrocephalus, HIV dementia, bipolar disorder, FTDP-17 (other tauopathies), Hepatic encephalopathy, or Lupus encephalitis.
33. The method of claim 31, wherein the modulation comprises administering to the subject a therapeutically effective amount of a calcium chelator.
34. The method of claim 33, wherein the calcium chelator is EDTA, EGTA, diazo-2, FURA- 2, Di-Bromo-BAPTA, or BAPTA-AM.
35. A method for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of at least one protein that participates in the expression or release of adenosine.
36. The method of claim 35, wherein the condition is convulsions.
37. The method of claim 35, wherein the protein is bradykinin receptors, mGluR5, P2Y1, P2Y4, P2Y2, or noradrenergic receptors.
38. The method of claim 35, wherein the proteins are SNARE proteins.
39. The method of claim 35, wherein the condition is convulsions.
40. The method of claim 35, wherein the modulation is inhibiting at least one gene encoding the protein.
41. The method of claim 40, wherein the inhibiting comprises administering to the subject an antisense nucleic acid, RNAi, or siRNA.
42. The method of claim 35, wherein the modulation comprises administering to the subject a therapeutically effective amount of a Clostridial toxin.
43. The method of claim 42, wherein the Clostridial toxin is tetanus toxin, botulinum toxin A, botulinum toxin B, botulinum toxin Cl, botulinum toxin D, botulinum toxin E, botulinum toxin F, or botulinum toxin G.
44. The method of claim 35, wherein the modulation comprises administering to the subject a therapeutically effective amount of at least one antagonist to at least one of the proteins.
45. The method of claim 44, wherein the antagonist inhibits formation of a SNARE complex.
46. The method of claim 44, wherein the modulating comprises administering to the subject a therapeutically effective amount of at least one agonist to at least one of the proteins.
47. The method of claim 46, wherein the agonist is OAG, DAG lipase inhibitor RHC-80267, glutamate, ATP, norepinephrine, acetylcholine, or bradykinin.
kinase.
49. A method for treating or preventing a neurological or psychiatric condition in a subject comprising modulating the expression or activity of at least one protein that participates in the expression or release of D-serine.
50. The method of claim 49, wherein the protein is the NMDA receptor.
51. The method of claim 49 wherein the condition is schizophrenia.
52. The method of claim 49, wherein the modulation comprises administering to the subject a therapeutically effective amount of at least one agonist to at least one of the proteins.
53. The method of claim 52, wherein the agonist is ACBD, homoquinolinic acid, D-serine, cycloserine, D-cycloserine, glutamate, ATP, norepinephrine, acetylcholine, or bradykinin.
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