METHODS FOR REDUCING OR INHIBITING BRAIN INFLAMMATION OR FOR
PROMOTING NEUROGENESIS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority under 35 U.S.C. §119 (e) from provisional U.S. application no. 60/622,679, filed October 28, 2004, the entire content of which is herein incorporated by reference.
BACKGROUND OF THE INVENTION Field of the Invention
[0002] The present invention relates to novel uses of an anti- amyloid beta (AJS) immunotherapy method aimed at reduction of brain inflammation as a result of microglia activation and promotion of neurogenesis, thus treating neurodegenerating conditions and at diseases like Alzheimer's disease, stroke, Parkinson's disease, glaucoma, brain injury, and others.
Description of the Related Art
[0003] Neurogenesis - the birth of new neurons within the central nervous system (CNS) , which continues throughout life - employs a cellular reservoir for replacement of cells lost during normal cell turnover and after brain injury. This process takes place especially in the sub-ventricular zone (SVZ) and in the dentate gyrus of the hippocampus. The amount of neurogenesis within the dentate gyrus correlates closely with hippocampal functions of learning and memory (Shors et al. , 2001; and Feng et al. , 2001) .
[0004] An increasing number of reports suggest that adult hippocampal neurogenesis is involved in hippocampal-mediated learning. Cognitive functions are known to be quantitatively correlated with hippocampal neurogenesis (Drapeau et al. , 2003) .
Indeed, the hippocampus is implicated in various forms of memory, and it has been shown that conditions that increase memory- performance, such as an enriched environment or running, and physical exercise, also enhance neurogenesis (Kempermann et al, 1997 and 1998; and van Praag et al . , 1999a and 1999b) . Conversely, situations that reduce neurogenesis, such as prenatal stress (Lemaire et al . , 2000) or an antimitotic treatment (Monje et al . , 2002) , have been associated with cognitive impairments.
[0005] Altogether, a higher neurogenesis was observed in animals with the best behavioral performances, indicating that a certain threshold of neurogenesis is required to maintain correct spatial memory.
[0006] The neurogenesis process involves stem cells originating from the ependima layer of the ventricles and in the hippocampus, which become either neuronal progenitor cells (NPC) or astrocytic progenitor cells (APC) and migrate to their final location and destiny. Along this process, they have to first of all survive and then become either neurons or astrocytes, depending on their microenvironment. Alterations in the microenvironment of the progenitor cell may allow ectopic neurogenesis to occur (Nakatomi et al. , 2002; and Magavi et al. , 2000) or even block essential neurogenesis, leading to deficits in learning and memory (Cameron et al. , 1998; Madsen et al . ; and Monje et al. , 2003) .
[0007] Alzheimer's disease (AD), as well as other neurodegenerating diseases in which neuronal loss is prominent, is accompanied with a shifting imbalance between neurogenesis and neurodegeneration, resulting in cell death, especially in the hippocampus. Nevertheless, the aged brain retains the capacity to up-regulate neurogenesis in response to physiological (Cameron et al . , 1999; and Kempermann et al. , 2002) and pathological (Gray et al., 2002) factors. Efforts to compensate for neuronal loss by
increasing neurogenesis has been documented in AD as well as in other neuropathological states, such as ischemia (Solway et al . , 1998) , which may represent a mechanism directed toward the replacement of dead or damaged neurons. However, neurogenesis does not fully compensate for neuronal loss in age-related neurodegenerative disorders.
[0008] Decreased neurogenesis is accompanied by activation of microglia, as neurogenesis is tightly correlated to the degree of microglial activation (Monje et al . , 2003) . Suppression of hippocampal neurogenesis by activated microglia may be one explanation of cognitive dysfunction, which adds a new angle to the rationale behind immunotherapeutics in AD.
[0009] While neurogenesis appears to be increased in the brains of patients with AD, progressive cell loss is still observed. This may be due to the disruptive microenvironment to neurogenesis in the AD brain, probably due to amyloid beta- peptide, which may be toxic to new neurons (Monje et al., 2003) . Amyloid beta-peptide (AjSP) , characteristically found in the plaques of brains with AD, is derived from the cleavage of amyloid precursor protein (Hardy et al . , 2002) . Previous studies showed that A/SP disrupts neurogenesis in the SVZ and hippocampus in mouse models of AD (Haughey et al. , 2002a), suggesting the possibility that the amyloid ^-peptide effect on impaired neurogenesis contributes to the pathogenesis of neurodegenerative disorders. The infusion of AjSP into the lateral ventricle of adult mice was also shown to impair neurogenesis in the subventricular region.
[0010] Neurogenesis is impaired in mice over-expressing a mutant form of APP that causes early onset autosomal dominant AD in humans (Haughey et al. , 2002b) . Recent studies of experimental models of AD have shown that A/3P can impair neurogenesis of neural progenitor cells. A1SP impaired
proliferation and neuronal differentiation of cultured human and rodent NPC,as well as of mouse NPC in vivo. A/3P also impaired the proliferation and neuronal differentiation of cultured human and rodent NPC, and promoted apoptosis of neuron-restricted NPC by a mechanism involving disregulation of cellular calcium homeostasis and the activation of calpains and caspases. Moreover, exposure of cultured human NPC to A/3P impairs their proliferation and differentiation and can induce apoptosis. The adverse effects of A/3P on NPC may contribute to depletion of neurons and cognitive impairment in AD (Mattson et al. , 2002 and 1992) . However, the status of neurogenesis in neurodegenerative disorders in humans is unknown.
[0011] Amyloid /3-peptide may be a beneficial short-term response factor in resolving extra-cellular stressors and in killing pathogens that have accessed nervous tissue and may play a physiological role in learning and memory; however, A/3P can become harmful when active over an extended period following ineffectual response to its persistent stimulus that cannot be cleared by activated microglia. There are probably additional possible independent mechanisms through which A/3 peptides may damage cells (Hertel et al . , 1996) . At nanomolar concentrations, A/3P renders the plasma membrane vulnerable to additional insults by non-selective interaction with lipids of the membrane (Hertel et al . , 1996), while at micromolar concentrations, A1SP may activate cell death pathways by activating a number of plasma membrane receptors and/or by induction of oxidative stress.
[0012] The use of an anti-amyloid beta immunotherapy method was previously shown to be efficient in preventing aggregation or in disaggregating amyloid plaques both in vitro (Frenkel et al . , 2000) and in vivo (Frenkel et al . , 2003; Solomon et al . , 2003; and Lavie et al . , 2004) .
[0013] Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods for reducing or inhibiting brain inflammation or for promoting neurogenesis following the onset of neurodegeneration in a neurodegenerative disease or disorder involving the pathological effects of amyloid beta, where a subject in a need thereof is administered by active or passive immunotherapy an effective amount of a phage displaying the amino acid sequence of EFRH (SEQ ID NO:1) as an epitope of amyloid beta or displaying a molecule having the antigen binding portion of an antibody specific for EFRH (SEQ ID NO:1) .
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figures Ia-Ic are graphs showing as follows: In Fig. Ia, i) improvement in spatial navigation performance of animal in the MWM test displayed as [100 x (1- path length at day 4/path length at day I)] : The anti-EFRH-treatment improved spatial navigation ability by about 30%, similar to that of non- transgenic young mice (44.5%), in contradiction to the deteriorated performance of control untreated transgenic mice (- 15%) . In Fig. Ib, alleviation of plague load: Mean plaque burden was measured in two sections -1.6 and -3.8 from bregma of the right hemisphere. Amyloid burden was calculated as percentage of the measured amyloid plaque area stained by thioflavin-S versus
the total section area. Results show a significant decrease of amyloid burden in treated compared to untreated Tg mice. In Fig. Ic, reduction in soluble A/3P1-40 (I) arid 1-42 (II) in the left hemisphere extracts: /3~amyloid content was measured as μg/gr brain wet weight. Both 1-40 and 1-42 AβPs levels were significantly reduced in treated compared to untreated Tg mice brains. Tg-transgenic mice; nTg-non transgenic mice; *show P<0.05(a) ; <0.015(b) ; <0.04(c) difference from control, untreated Tg animals.
[0016] Figure 2 shows anti-EFRH immunization effects on brain inflammation.
[0017] I. F4/80 staining of activated microglia in mice brains showed significant effect of the treatment in reducing brain inflammation. a. Untreated Tg animals: Labeling depicts many ramified, activated microglia (thin arrows) within the hippocampus. Labeling is also surrounding blood vessels (thick arrows) and amyloid plaques, (starlet) b. Treated Tg animals: only sparse labeling can be found.
Scale bar in (b) (50mμ) corresponds also to (a) .
II. F4/80 scores show highly significant decreases in labeling in the treated compared to untreated animals. Tg-transgenic mice; nTg-non transgenic mice; * show <0.00003 difference from control, untreated Tg animals.
[0018] Figure 3 shows anti-EFRH immunization effect on neuronal density and neuronal cell size in the hippocampus. Neurons, stained by the neuronal marker NeuN, were counted and their size measured in two areas of CAl and CA3 and in the granular layer of the dentate gyrus (DG) at the level of -1.2 - - 1.4 from bregma. I. Schematic representation of the areas measured. II a. -Neuronal density was defined as the number of
NeuN+ neurons in mm2 of the cellular layer and shown to be reduced in Tg untreated animals compared to nonTg animals, but not in the treated animals (* p<0.04 from Tg untreated; + p<0.03 from nTg controls) II b. -Neuronal cell size, obtained from measurements of thionin staining of the same areas, was significantly reduced in Tg control mice compared to nTg mice, but not in treated animals (* p<0.05 from Tg untreated; + p<0.05 from nTg controls) .
[0019] Figure 4 shows doublecortin+ cell distribution and morphology.
[0020] I. DCX+ CeIIs distribution. a,b.- Representation of DCX+ cells distribution in Tg mice brain of treated (a) vs. untreated (b) animals. Section images were density sliced and labeled cells were color coded red. Note the wide distribution of DCX+ cells in the treated animals. Cx-Cortex; Hip-hippocampus; Hillus- hillus; Thai-thalamus; ic-internal capsule;Amyg- amygdala; Scale bar in (b) corresponds also to (a) .
[0021] II. DCX
+ morphology a,b.- DCX
+ cells in the dentate gyrus of treated vs. untreated Tg mice. In the treated animal, DCX
+ giant multi-processed cells are interwoven with the neurons of the granular cell layer. c,d.- DCX
+ cells, adjacent to the ependima of the lateral ventricle (LV) (c) and the olfactory ventricle (OV) (d) . e.- DCX
+ cells in the striatum are in close proximity to blood vessels (bv) . f.-DCX
+ cells with bipolar morphology resemble migrating neurons in the striatum (g) . DCX
+ in CA
3 (h,i) . DCX
+ cells surrounding plaques in treated (h) but not in untreated animals (i) Plaques are characteristically dusted with DCX
+ cell fragments and distrophic neurites(i) . Scale bar in g corresponds also to
Scale bar in i corresponds also to h.
[0022] III. DXC+ Cell density. Mean number of cells/mm2 in each area of brain section: No DCX+ cells were observed in non-
transgenic control animals. Only a few cells, mostly in the hippocampus, were found in untreated transgenic animals. In treated animals DCX+ cells were more numerous and had a wider distribution.
[0023] Figure 5 shows DXC+ cells colocalize with Tuj-1 and/or with GFAP.
[0024] I. Colocalization of DCX+ with the neuronal marker Tuj-1 in different areas of the brain: DCX-red; Tuj -1-Green; DAPI-Blue; a-d.- The pyramidal cell layer and the stratum radiation of hippocampus, e-g.- granular cell layer of the dentated gyrus, h. -cortex; i. -amygdala, j.-hillus, Big arrows indicate DCX+/TUJ-1+ labeled cells. Small arrows indicate DCX+ cells. a,e.- DCX staining, b.f.- Tuj-1 staining, c.-DAPI staining, d.-a+b+c merged, e. -j . -Confocal images. e.-DCX+ only; f. -Tuj-1 only g.-e+f merged. g-j . -DCX+/TUJ-1+ cells: Doublecortin labeling surround the unlabeled nucleus and extend, compared to the Tuj-1, to only a limited distance into the cell's processes. Scale bar in c - 50μm corresponds also to a,b; . Scale bar in d-50μm; Scale Bar in j-20μm corresponds also to e,f,I,g; Scale bar in h-5μm.
[0025] II. DCX+ Colocalizes with GFAP in the brain: DCX-red; GFAP-Green; DAPI-Blue: a. -DCX staining b.-GFAP staining; c.-DAPI staining, d. -a+b+c merged-in the hippocampal hilus: . DCX+/GFAP+ (yellow, big arrows) among many astrocytes and astrocytic processes (green, small arrows) and DCX+ only cells (red, arrow heads) . e-j . -Confocal image. β/h/k.-DCX staining; f,i,l.-GFAP staining; g,j ,m. -merged. DCX granular material surrounds the unlabeled nucleus and, compared to the GFAP, extends only a limited distance into the cell's processes, k-m show a very big DCX+/GFAP+ cell adjacent to an amyloid plaque (asterix) in an untreated animal . The amyloid plaque contains GFAP+ distrophic neurites and is surrounded by DCX+ cell processes/fragments.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In many circumstances, A/3P appears to act in a cytokine-like manner and promote inflammatory events. Interestingly, AjS deposition, vascular damage, and the resultant gliosis occur together, and /3-amyloid 1-40 has been shown to increase vasoconstriction only due to soluble A/3P more than to its known toxic amyloid plaques. The data presented in the example hereinbelow show that immunotherapy resulting in a minimal titer against EFRH (SEQ ID NO:1) was enough to alleviate neuroinflammation, amyoid burden and lowered soluble forms of A/3P. The treatment was highly effective on animal behavior in the MWM, which was as good as that of normal, younger mice. This effect, resulting from the treatment, was correlated with the low titer against EFRH (SEQ ID N0:l) .
[0027] It is believed that the beneficial effect of the immunotherapy might stem less from alleviation of the toxic plaques and more so from reduction of brain inflammation and promotion and/or restoration of neurogenesis, all resulting in normal numbers of functioning neurons.
[0028] Neurogenesis occurs in the adult mammalian brain and may play roles in learning and memory processes and recovery from injury. AD, as well as other neurodegenerative diseases, is accompanied by shifting imbalance between neurogenesis and neurodegeneration, resulting in cell death, especially in the hippocampus.
[0029] While neurogenesis appears to be increased in the brains of patients with AD, progressive cell loss is still observed. This may be due to the disruptive microenvironment to neurogenesis in the AD brain which may be toxic to new neurons (Jin et al., 2004) due to brain inflammation. Brain inflammation causes inhibition of neurogenesis both in the basal continuous
formation of new neurons in intact hippocampal formation and in increased neurogenesis in response to a brain insult. Impairment of neurogenesis depends on the degree of microglia activation, irrespective of whether there is damage or not in the surrounding tissue.
[0030] Brain inflammation probably plays an important role in the pathogenesis of other chronic neurodegenerative disorders besides AD, which involves A/3P pathological effects, like Parkinson's disease, Lewy Body Dementia, AIDS Dementia Complex, traumatic brain injury, glaucoma, etc.
[0031] Glaucoma is a chronic neurodegeneration of the optic nerve and one of the leading causes of vision loss in the world among the aging. Retinal ganglion cells (RGCs) have been shown to die by apoptosis. Central to apoptosis is the activation of caspases which are activated also in other chronic neurodegenerations as well as in AD and after optic nerve transection. It was previously shown in rat glaucoma models that caspase-3 and caspase-8 are activated in RGCs and cleave amyloid precursor protein (APP) to produce neurotoxic fragments that include amyloid-beta (McKinnon et al. , 2003) . This suggests a new hypothesis for RGC death in glaucoma involving chronic amyloid- beta neurotoxicity, mimicking AD at the molecular level . The benefits are that treatments for AD could be used to treat glaucoma, and strategies developed to treat glaucoma could treat other neurodegenerations.
[0032] Traumatic brain injury (TBI) includes /3-amyloid deposition and the early onset of dementia. Despite such descriptions, little is known about the mechanisms through which these changes occur. One source proposed for the generation of /3-amyloid peptide in TBI is abnormal proteolytic cleavage of the /3-amyloid precursor protein (APP) that has been shown to accumulate at sites of impaired axoplasmic transport within
traumatically injured axons. In fact, jSA immunoreactivity has recently been found with swollen axons in a pig model of TBI (Smith et al . , 1999), suggesting the accumulation of APP in traumatically injured axons may be a source for /3-amyloid peptide formation in TBI .
[0033] The present invention provides a method for reducing or inhibiting brain inflammation in a neurodengerative disease or disorder involving the pathological effects of amyloid beta, where a subject in need thereof is administered by active or passive immunotherapy an effective amount of a phage, preferably a filamentous phage, displaying the amino acid sequence of EFRH (SEQ ID NO:1) as an epitope of amyloid beta or displaying a molecule having the antigen binding portion of an antibody specific for EFRH (SEQ ID NO:1) to control microglial activation, thereby reducing or inhibiting brain inflammation.
[0034] The present invention further provides a method for promoting neurogenesis following the onset of neurodegeneration in a neurodegenerative disease or disorder involving the pathological effects of amyloid beta, where a subject in need thereof is administered by active or passive immunotherapy an effective amount of a phage, preferably a filamentous phage, displaying the amino acid sequence of EFRH (SEQ ID N0:l) as an epitope of amyloid beta or displaying a molecule having the antigen binding portion of an antibody specific for EFRH (SEQ ID NO:1) to promote neurogenesis and thereby counterbalance the neurodegeneration.
[0035] Non-limiting examples of neurodegenerative diseases and disorders include Alzheimer's disease, Parkinson's disease, Lewy Body Dementia, AIDS Dementia Complex, stroke, traumatic brain injury, and glaucoma.
[0036] The phage displaying EFRH (SEQ ID N0:l) or displaying a molecule having the antigen binding potion of an antibody
specific for EFRH (SEQ ID NO:1) is known in the art as disclosed in U.S. Patent 6,703,015, the entire contents of which are hereby incorporated by reference.
[0037] Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration and is not intended to be limiting of the present invention.
EXAMPLE
[0038] Neurogenesis is currently of intense interest and extensive research but stands in the midst of bitter debate concerning ethical and practical problems. Neurodegenerating diseases such as Alzheimer's are accompanied with shifting imbalance between neurogenesis and neurodegeneration, resulting in cell death attributed to the over-expressed amyloid beta peptide (AjSP) neurotoxicity. The laboratory of the present inventors have previously shown that antibodies against the EFRH sequence of A/3P prevent aggregation and disaggregate A/3 both in vitro and in vivo. EFRH, located at the soluble tail of the N- terminal region, acts as a regulatory site controlling both the solubilization and disaggregation process of the AβP molecule. In this example, it is shown that anti-EFRH immunotherapy of hAPP transgenic mice promoted neurogenesis, expressed by enhanced numbers of doublecortin (DCX) labeled cells multiplied in various regions of the brain. The treatment reduced brain inflammation and alleviated amyloid load, which probably led to the improvement in behavior in the Morris Water Maze task (MWM) . Moreover, neuronal density in the cellular layers of the hippocampus was significantly higher in treated compared to untreated animals and resembled that of normal non-transgenic mice, thus explaining the improvement in cognition functions. In untreated animals, almost all DCX+ cells colocalized with GFAP,
while in treated mice DCX+ labeled cells colocalized either with GFAP or with Tuj-1. These findings suggest that amyloid immunotherapy not only prevents neurodegeneration but may also promote recovery from Alzheimer's disease (AD) or other diseases related to A/3P overproduction and neurotoxicity, and might restore neuronal as well as cognitive functions of treated patients.
[0039] Abbreviations: DCX, doublecortin; A/3P, amyloid beta peptide; APP, amyloid precursor protein; MWM, Morris Water Maze; Tg, hAPP transgenic mice; Tuj-1, Neuron specific beta III Tubulin; GFAP, glial fibrillary astrocytic protein;
MATERIALS AND METHODS
[0040] Mice. The experiments were performed on transgenic mice which express human APP75I regulated by the neuronal murine Thyl promoter. The hAPP gene carries both London (717) and Swedish (670/671) mutations, resulting in an age-dependent increase in A/3. Over-expression of mutated human APP results in the development of typical AjS depositions as amyloid plaques in the neocortex and hippocampus .
[0041] Antigens. Filamentous phages were engineered to display the sequence EFRH (SEQ ID NO:1) , located between amino acid residues 3-6 of A/3P, on either their minor or major coat proteins using molecular techniques. Two types of phages were constructed and used for immunization of the animals: 'a' is a phage displaying an EFRHEFRH (SEQ ID NO:2) sequence (a tandem repeat) on the minor coat protein P3 of the phage, and because every phage particle harbors 5 copies of P3, BS-I displays approximately 10 copies of the EFRH (SEQ ID NO:1) epitope, 'b1 has the sequence EFRHEFRH (SEQ ID NO:2) displayed on each of the recombinant P8 proteins of the phage, bringing the number of EFRH (SEQ ID NO:1) copies presented by these phages to approximately
300. Single chain antibodies (scPv) were raised against the same epitope and were displayed on filamentous phage (Frenkel et al. , 2000b; Cohen-Kupiec et al- , 2005)
[0042] Intranasal phage-ScFv vaccination. In addition, phage displaying ScFv raised against the same sequence of EFRH (SEQ ID N0:l) was used for passive immunization via intranasal administration (Frenkel et al. , 2002) .
[0043] Nine to ten month old hAPP transgenic mice (Tg) mice were immunized with the EFRH antigens administered intraperitoneally (i.p.) (n=13) as previously described (Lavie et al . , 2004) , or with anti-EFRH antibody displayed on phages administered intranasally (i.n) (n=9) by the same regimen. Six intraperitoneal (i.p.) injections of 1011 phages/mouse took place every 3 weeks for a total period of 12 weeks. The scFv was administered similarly, but via intranasal administration.
[0044] At the end of this period, the immune response of the animals was measured using ELISA. Wells of microtiter plates (Maxisorb, Nunc) were coated with 20 μg avidin in 0.1 M NaHCO3, pH 8.6, and incubated overnight at 40C. Biotinylated beta-amyloid peptide 1-16 at 1 μg/ml in PBS was added for 30 nain. at room temperature. PBS containing bovine serum albumin (1.5 mg/ml) was used to block all non-specific binding sites for 2 hr at 370C. After each step the wells were rinsed three times with PBS containing 0.05% Tween-20. Diluted mice sera were incubated for lhr at 37°C and the binding of anti-EFRH antibodies was measured as described above.
[0045] Behavioral Tests. The Morris water maze (MWM) test was used at the end of the immunization treatment to examine behavioral differences between the test and control mice groups. In the trials, the escape latencies, length of swimming path, swimming speed and abidance in each quadrant of the pool were measured and analyzed for each animal . Performance was evaluated
by testing the spatial and temporal navigation abilities of each animal. The animals included in the study have dark eyes and were able to perfectly perceive the landmarks outside the MWM pool . In an MWM trial, a mouse swims to find a hidden platform, using visual cues. The task is based on the principle that rodents are highly motivated to escape from a water environment by the quickest, most direct route. This task is carried out in a circular pool (Im diameter) filled with 23 +20C warm water and virtually divided into four quadrants. Swimming path lengths were measured and analyzed for each animal. During each trial, the mouse is allowed 60 seconds to reach the platform. The location of the platform in the pool does not change; however, the trial starting point changes randomly using a computer. Each animal performs 3 training trials per day, with 10 minute inter- trial intervals for a total of 4 days. A spatial probe trial (60 seconds) is given one hour after the last training trial on day 4. The effect of the treatment is displayed here as the improvement in the animal ' s performance over the 4 days of the trial. The animal behavior improvement is thus calculated as 100 x (1- path length at day 4/ path length at day 1) .
[0046] Preparation of brain tissue. The hAPP mice were euthanized with chloroform and immediately decapitated. After brain removal, the right cerebral hemisphere was post-fixed for 24 hours in 4% paraformaldehyde/PBS (pH 7.4) . The left cerebral hemisphere was frozen on dry ice for further biochemical analysis (A/3 determination) .
[0047] The right, fixed cerebral hemisphere was embedded in paraffin. Serial coronal sections (5μm) , 250μm apart, were prepared for histology.
[0048] Quantitative analysis of amyloid plaque load. Coronal sections were cut from the occipital two-thirds of the right hemisphere. Quantitative analysis of vascular and plaque amyloid
in the brain was performed after thioflavin-S staining. Two well-defined coronal sections at the levels of -1.6 and -3.6 from bregma, respectively, were selected for quantification of the amount of amyloid load. Images from these sections were collected from a CCD color video camera (ProgRes C14, Jenoptic, Jena) and analyzed with appropriate software (Leica Qwin, Leica) . The total amyloid dense core load in plaques was expressed as a percentage of the area stained with thioflavin-S of the total area of the section.
[0049] Determination of AβP. After brain removal, the left cerebral hemisphere was frozen on dry ice for A/3 determination which was carried out as follows: Samples out of the TRIS buffered saline (TBS) as well as of the formic acid brain preparations were analyzed in both, an A/3P 1-40 ELISA as well as an AjδP 1-42 ELISA (from Abeta®, Germany) . In brief, frozen hemispheres were homogenized in TBS-buffer (5 ml) containing protease inhibitor cocktail. After centrifugation, the supernatants were aliquoted and kept at -200C. The pellets were homogenized in 70% formic acid (ImI) prior to centrifugation. The supernatant obtained from centrifugation was neutralized with IM TRIS (19ml) and used for ELISA.
[0050] With this amyloid-^ test-kit, the principle of a solid phase enzyme immunoassay, an Enzyme-Linked-Immunsorbent-Assay (ELISA) , for the quantitative analysis of hA/3P 1-40/hAβP 1-42 was applied. The antigen hA/3P l-40/hA/3P 1-42 to be tested was detected by selective monoclonal anti-AjS antibodies at two different binding sites.
[0051] Antibodies. Guinea pig α-DCX (1:500) and goat α-guinea pig IgG Cy3 (1:200; Chemicon International, Temecula, USA), mouse ce-Tuj-1 (1:500; R&D Systems Inc. Minneapolis, USA), rabbit α-GFAP (1:1000,- DAKO Cytomation, Glostrup, Denmark) , mouse anti-NeuN (1:400), Gα-rabbit-biotin (ready to use) , histomouse kit, broad
range polymer-HRP from the Picture Plus kit; diaminobenzidin (DAB) and DABm from Zymed Laboratories Inc. South San-Francisco, USA; goat α-guinea pig HRP (1:300), goat .α-mouse-Cy2 (1:500) and Avidin-Fitc (1:300) from Jackson Immunoresearch Laboratories, West Grove, PA, USA; α-mouse F4/80 (1:100; Serotec, Oxford, UK), ultra V block (ready to use; Labvision, Fremont, CA, USA) .
[0052] Iπanunolabeling. Immunohistochemical labeling was performed on serial coronal paraffin sections (5 μm) 250 μm apart from each other. The sections were deparaffinized by a series of xylenes, hydrated with a gradient series of ethanols and quenched by 3% H2O2 in methanol. For visualization with diaminobenzidin (DAB) all sections were quenched by 3% H2O2 in methanol, blocked by Histomouse kit Blocker A, and further processed for immunolabeling. Antibodies were visualized with broad range polymer HRP from the Picture Plus kit (unless otherwise specified) , reacted with DAB and counterstained with heamatoxylin. Microscopic evaluation was done using Leica DMLB microscope. For visualization with fluorescent second antibodies, antigen retrieval was performed for 20 min in Tris-EGTA buffer 0.01M; pH-9.0.
1) Doublecortin
[0053] Doublecortin, a neuronal marker, is transiently expressed in progenitor neuronal cells involved in neuronal migration through Ca2+-dependent signaling. It is a component of the microtubule cytoskeleton and is essential in postmitotic neurons as it stabilizes microtubules and stimulates their polymerization (Friocourt et al . , 2003) . Immunolabeling with doublecortin suggests generation of new cells in the brain.
[0054] Blocking was done first by Histomouse kit Blocker A (Zymed) for 30 min at room temperature followed by 10%NGS/3%BSA/0.1%Triton/TBS for 20 min at room temperature. Sections were then incubated for 2h at room temperature with
guinea pig anti-DCX antibody (Chemicon, 1:500) , washed with TBS and incubated for Ih with goat anti-guinea pig HRP (Johnson Laboratory Research 1:300) . Immunolabeling was visualized by Diaminobenzidin (Zymed) for 5 min. Sections were counter-stained with heamatoxylin, dehydrated, cleared and mounted. Microscopical evaluation was done using a Leica DMLB microscope.
2) F4/80
[0055] Blocking was done first by Histomouse kit Blocker A (Zymed) for 30 min at room temperature followed by 3% non-fat milk in TBS. Sections were then incubated overnight at 40C with rat anti-mouse F4/80 (Serotec 1:100) in TBS. Antibody was visualized using a broad range polymer-HRP (Picture Plus, Zymed) , developed with DAB. F4/80 labeling was evaluated by a blinded-to- the-treatment investigator using a Leica DMLB microscope at a magnification of x630 and scored between 1-10 according to the labeling density. In each brain, the cortex, hippocampus, thalamus and striatum areas were scored separately and the final score was the sum of each of these scores.
3) Immunofluorescent double-labeling of DCX- with Tuj-1 or GFAP
[0056] Blocking was carried out with 10% normal goat serum/3%BSA/0.01M in PBS/O.1% Triton followed by Ultra V block. The sections were incubated with α-DCX overnight at 40C followed by 4h at RT and visualized by goat anti-guinea pig IgG Cy3. For double-labeling, sections were incubated with mouse α-Tuj-1 visualized by Gα-mouse-Cy2 or with rabbit α-GFAP visualized by
Gα-rabbit-biotin followed by Avidin-FITC. Microscopic evaluation was also performed with LS510 Ziess confocal microscope.
4) NeuN
[0057] Antigen retrieval was performed for 3 min twice in citric buffer pH-6.0. Blocking was done by UV block for 5 min at RT. Sections were then incubated overnight at 4°C with mouse anti-mouse NeuN in PBS.
[0058] Quantitation of iπαnunolabeling. All measurements were done by a blind-to-the-treatment investigator.
Doublecort±n expressing (DCX+) cells density
[0059] DXC+ cells were counted in all the sections (6-9 for each animal) using the microscope at a magnification of x200 and their density was calculated as number of cells/mm2 of brain section. Only those DCX+ cells with a visible nucleus were counted.
F4/80 score
[0060] F4/80 labeling was examined using the microscope at a magnification of x630, scored between 1-10, according to labeling intensity. In each brain, the cortex, hippocampus, thalamus and striatum areas were scored separately and the final score was the sum of each of these scores.
Hippocampal NeuN expressing (NeuN+) cells density and size
[0061] NeuN+ cell density was measured by stereological method in two fields of 0.13 mm2 alongside each of the CAl, CA3 cellular layer of the hippocampus, and the granular cell layer of the dentate gyrus (Fig.3 1) . The NeuN - a marker specific to adult neurons - was used to avoid the possible mistake of counting non-neuronal cells rather than neurons only. The stereological method was also used for measuring the neuronal cell size. For that purpose we used thionin staining.
RESULTS
[0062] Improvement in cognitive functions. The treatment affects learning and memory, as demonstrated in the MWM test (Lavie et al. , 2004) . After the anti-EFRH-treatment, the performance (spatial navigation) of treated mice improved by about 30%, similar to that of young non-transgenic mice (44.5%), and contradictory to the performance of untreated transgenic mice (-15%) (Fig. Ia) .
[0063] .Reduction of amyloid burden. Neuropathology analysis of the total amyloid burden in the right hemisphere, measured following thioflavin-S staining (Fig.Ib) , and of soluble A/3P 1-40 (Fig.Ic I) and 1-42 (Fig. Ic II) contents in the whole left hemisphere extracts show a significant decrease of amyloid burden in brains of treated mice compared to untreated Tg mice.
[0064] Reduced brain inflammation. Brains of Alzheimer's diseased patients, and of hAPP Tg mice serving as animal models for Alzheimer's disease, are characterized by proliferation and activation of microglia manifesting inflammation which is considered to be related to the amyloid plaques (Ekdahl et al . , 2003; and Perry et al . , 2003) . The treatment effect on brain inflammation was evaluated by immunohistochemistry staining with F4/80, a marker of reactive microglia (Lawson et al. , 1992) . The treatment significantly reduced brain inflammation, as shown in (Fig.2) .
[0065] Restoration of density and size of adult NeuN labeled neurons in the cellular layers of the hippocampus. The density of adult neurons expressing the neuronal marker NeuN and on neuronal size in the CAl, CA3 and dentate gyrus of the hippocampus was evaluated (Fig.3 I) .
[0066] Compared to nTg animals, neuronal cells density was reduced significantly in Tg control animals but restored in the treated animals (1896+97; 1447+62; 1765+ 41 in CAl, 1495+ 129;
1142+_ 51; 1499+_ in CA3 and 3291+ 383; 3208+96; 3530+222 in the DG of non- Tg, Tg control and Tg treated animals respectively) (Fig. 3 II a) . Neuronal cell size, obtained from measurements of thionin staining of the same sections (Fig. 3 II b) , was significantly reduced in Tg control mice compared to nTg mice, which is reversed in treated animals (25.0+_5.95 18.10+1.27; 21.81+_ 0.88 in CAl, 26.38+ 6.84; 16.55+ 1.31; 21.52+1.44 in CA3 and 31.90+_ 1.58; 24.3+1.39; 31.15+3.13 in the DG of nTg, Tg control and Tg treated animals, respectively) .
[0067] Promotion of neurogenesis: Doublecortin expressing cells in treated/untreated Tg mice. DCX+ cells in areas like the olfactory nuclei, piriform cortex, supracallosal areas of the cortex, amygdala, striatum and the thalamus were measured. They are numerous in the hippocampus, especially in the granular and subgranular zone of the dentate gyrus, the hilus and the subiculum. DCX+ cells also reside in white matter neighborhoods like the corpus callosum, especially in the forceps minor, the anterior commisure, fimbria, the stria of the striatum, the internal capsule, the optic tract, the cerebral peduncle and in other areas. In these areas they usually reside in close proximity to blood vessels. Only a small number of cells were detected in transgenic untreated animals, mainly in the hippocampus. Fig.4, I a,b, shows DCX+ cell distribution in treated compared to untreated brains.
[0068] DCX+ labeled cell morphology can be divided into two types: giant, multipolar cells resembling astrocytes, and bipolar cells bearing a small number of processes resembling neurons. Both types have a large, pale unlabeled nucleus with a few nucleoli. Fig. 4,11 shows DCX+ cell morphology: typical multipolar DCX+ cells residing in the subgranular and granular layer of the dentate gyrus in which they are interwoven with neighboring neurons (a) . Their nuclei are similar to the GCL
neuronal nuclei. Such cells are not detected in the untreated animal (b) . DCX+ cells are found adjacent to the ependima layer of the lateral and olfactory ventricles (c and d) . In white matter areas, many DCX+ cells are in close proximity to blood vessels (e) . Bipolar cells, resembling migrating neurons, are shown in the striatum (f) . DCX+ labeled neurons are shown among the CA3 of the hippocampus (g) . In certain areas rich in amyloid plaques, like the supracallosal areas of the cortex and the hippocampus, DCX+ cells, mainly of the giant multipolar type, are found to surround the plaques (h) . Plaques in other areas, like the upper layers of the cortex and in untreated animals, are devoid of them but are characteristically dusted with DCX+ cell fragments (i) .
[0069] DXC+ cells were counted in different areas of the brain and their density was calculated as the mean number of DCX+ cells in each area/mm2 of brain section. A small number of cells were found in the transgenic untreated animals, mostly in the hippocampus. In the treated transgenic animals, DCX+ cells were widely distributed and their density was much higher, suggesting that the treatment promotes both generation and migration of the DCX+ cells to different brain regions.
[0070] Double-labeling- of DCX with Tuj-1 and GFAP. DCX+ progenitor neuronal cells resemble both neurons and astrocytes. To analyze their nature, they were double-labeled them, either with Neuron specific beta III Tubulin (Tuj-1), a neuronal marker, or with the glial fibrillary astrocytic protein (GFAP) , an astrocytic marker.
[0071] DCX+ cells which colocalized with Tuj-1 have bipolar neuronal-like morphology. Fig.51 depicts such cells in the pyramidal cell layer and in the stratum radiation of the hippocampus (a-d) , the cortex (e) and the amygdala (f,g) . Confocal microscopy confirmed doublecortin colocalization with
Tuj-1 in the granular cell layer of the dentate gyrus (h-j) . Image in (g) was rotated in the orthogonal planes (x, y, z) to verify the double labeling.
[0072] DCX+ cells, which colocalize with GFAP, are the multipolar giant cells (Fig.5.II) . DXC+/GFAP+ cells are a fraction of the astrocytes, GFAP+ cells, population found adjacent to each other (Fig.5.II d) . Confocal microscopy confirmed doublecortin colocalization with GFAP (e-m) . Images in (g) and (j) were rotated in the orthogonal planes (x, y, z) to verify the double-labeling.
[0073] Many of the DCX+/GFAP+ cells surround amyloid plaques. In untreated animals, these cells frequently send processes deep into the amyloid plaque (Fig.5.II k-m) , while in treated animals they avoid it.
[0074] Comparison of DXC+/GFAP+ double-labeling between the studied animals shows that in transgenic untreated animals almost all DCX+ cells colocalized only with GFAP and are multipolar cells. In contrast, in treated animals, many DCX+ labeled cells colocalized with Tuj-1 and are bipolar.
DISCUSSION
[0075] Neurogenesis in the adult brain is now a well- recognized phenomenon. Demonstration that adult neurogenesis can be modulated by environmental factors holds good prospects for a variety of neuronal-replacement therapies. Neurogenesis is modulated by several normal and pathologic conditions, suggesting the involvement of the hippocampus and the subventricular zone in a broad range of functions, and that environmental stimuli and pathological conditions may have long-term consequences on the architecture and functioning of the central nervous system (Altman et al . , 1965; Kuhn et al . , 1996; Kempermann et al . , 2003; Powrozek et al . , 2004; Taupin, 2005; and Sugaya, 2005) .
[0076] In this Example, anti-EFRH immunization of transgenic mice is shown to reduce amyloid load and brain inflammation. Most importantly, the treatment promoted "self"-neurogenesis, expressed by enhanced numbers of DCX labeled cells multiplied in various regions of the brain. These cells originate from an inner reservoir of stem cells and migrate to different brain regions. Neuronal cell density in various regions of the hippocampus was elevated, probably supporting the improvement in cognitive behavior.
[0077] DCX+ cells were previously demonstrated to reside mostly in the SVZ, producing new neurons which migrate along the rostral migratory stream and in the subgranular zone at the dentate gyrus (Kuhn et al . , 1996; and Lois et al. , 1993, 1994 and 1996) but also in other areas including white matter (Nacher et al . , 2001) and surrounding blood vessels (Doetsch, 2003; and Shen et al. , 2004) . Magnetically labeled multipotential neural precursor cells, transplanted into the ventricles of rats with acute experimental autoimmune encephalomyelitis, migrate via white matter structures (Bulte et al. , 2003; and Ben-Hur et al . , 2003) . Those cells may mature and become functional neurons when incorporated into healthy brain (Song et al . , 2002; and van Praag et al. , 2002) .
[0078] The results here showed that the neuronal density in the cellular layers of the hippocampus was significantly higher in treated compared to untreated animals and resembled that of normal non-transgenic mice, probably as a result of the newly born neurons being properly incorporated, thus contributing to the treatment effect on improvement in cognition functions.
[0079] In this study, only a small number of DXC+ cells were detected in transgenic untreated animals, mainly in the hippocampus, while the anti-EFRH treatment resulted in a multiple number of DCX+ cells in many areas of the brain manifesting
induction of or recruiting an inner reservoir of stem cells for self-neurogenesis. These cells fell into two main categories: astrocytic-like and neuronal-like which expressed the glial marker GFAP and the neuronal marker Tuj-1, respectively. It seems that colocalization of DCX with GFAP marks a special stage in the development of neuronal progenitor cells, which might then be induced to become colocalized with Tuj-1 upon maturation to adult neurons. Otherwise, these cells may be arrested, continuing to express GFAP, or die upon encountering the neurotoxic plaques. Indeed, in untreated animals, almost all the DCX+ cells colocalize with GFAP, while in the treated animals many of them colocalize also with Tuj-1. This sequence of events may explain why many GFAP+ and GFAP+/DCX+ cell fragments manifesting cell death were found in the plaque area in untreated animals, but not in treated animals .
[0080] The identification of astrocytes as stem cells in adult mammals is now drawing attention and raises the fascinating possibility that glia throughout the brain, or a subset of them, may be latent stem cells (Doetsch, 2003) .
[0081] A/3P, either in vitro or in vivo, could exert its toxic effects on a broad population of susceptible neurons and non- neuronal cells via various mechanisms (Esteban, 2004; DeKosky, 2003; and Rowan et al. , 2003) .
[0082] In AD, astrocytes are thought to play a protective role by shielding neurons from the toxic effects of extracellular senile plaques. Recent work has shown that astrocytes that become reactive expressing GFAP, migrate in response to chemotactic stimuli, become immobilized when they encounter A/3 and, in response, produce cytokines and chemokines. Once in the presence of A/3, astrocytes can internalize and degrade it (Dong et al . , 2001; and Wyss-Coray et al . , 2002 and 2003) (for review see (Guenette, 2003) . Indeed, in untreated animals in this study,
many GFAP+ and DXC+/GFAP+ cells which clustered around the senile plaques and sent their processes deep into them were observed, while the plaques themselves contain GFAP+ cell fragments. In treated animals the plaques are almost devoid of cell fragments (Fig. 5 II) .
[0083] Recent studies point towards the beneficial effect of the anti-EFRH treatment, as immunotherapy was demonstrated to neutralize A/3 oligomers that disrupt the synaptic plasticity correlated with learning and memory (Klyubin et al . , 2005) . In addition, the beneficial effect of the anti-EFRH treatment might stem from the neurogenic effect of "soluble" forms of A/3P (Lopez- Toledano et al. , 2004) dissolved by the treatment from the previously aggregated dense plaques. The dependence of the neurogenic effect of A/3P on the state of aggregation of AjS suggests that the formation of new neurons is more likely to be induced by the "soluble" forms of A/3P than by the A/3 that has been organized into senile plaques. One possible interpretation of these data is that in earlier stages of Alzheimer's disease, when the excess of A/3P is enough to form oligomeric but not fibrillar aggregates, the oligomers (Dahlgren et al . , 2002; and Kim et al. , 2003) could activate a compensatory mechanism to replace lost or damaged neurons by increasing the differentiation of neuronal progenitors into new neurons. As time passes and senile plaques are formed, the balance could shift to fibrillar A/3 that could be more neurotoxic.
[0084] A(S, upon accumulation, activates non-neuronal cells, such as microglial and/or astroglial cells, which results in the production of neurotoxins and free radicals, leading to an increase of oxidative stress. The anti-EFRH treatment reduced the amyloid load thus alleviating the neurotoxic effect of amyloid plaque and reduced brain inflammation manifested by the significant reduction in F4/80 labeling. In AD patients' brains,
neurogenesis appears to be increased but progressive cell loss is still observed probably due to disruption of the micro environment necessary for neurogenesis as a result of brain inflammation (Ekdahl et al. , 2003; Perry et al . , 2003; and Monje et al., 2003) . Brain inflammation causes inhibition of neurogenesis, both in the basal continuous formation of new neurons in intact hippocampal formation and in increased neurogenesis in response to a brain insult. Impairment of neurogenesis depends on the degree of microglia activation, irrespective of whether there is damage or not in the surrounding tissue. The deleterious effect of activated microglia on the newly formed neurons is most likely mediated through the action of cytokines, such as IL-1/3 or IL-6, tumor necrosis factor oι, nitric oxide, and reactive oxygen species that can be released from microglia and are neurotoxic in vitro.
[0085] It was previously shown that neurogenesis is hampered by neuroinflammation and that precursor cells will rather become astrocytes than neurons at conditions of chronic inflammation (Sugaya, 2005a and 2005b) . For this reason, the anti-EFRH immunization, which significantly reduced brain F4/80 scores in treated compared to untreated animals, may promote generation of neuronal precursor cells to lead them towards maturation. This argument is supported by our results showing that most of the DCX+ cells in the untreated animals colocalized with GFAP, while in treated animals the DCX+ cells density was multiplied and many of them were colocalized with Tuj-1.
[0086] The beneficial effects of the anti-EFRH treatment are consistent with the model in which inflammation-mediated suppression of hippocampal neurogenesis plays a pathophysiological role for the cognitive dysfunction in these conditions, as the treatment blocked the detrimental effects of the amyloid plaques and restored neuronal density in the
hippocampus and cognitive functions. Moreover, it is demonstrated here that the anti-EFRH immunization, which alleviated the neurotoxic effect of amyloid plaque and reduced brain inflammation, promoted neurogenesis and allowed the DCX+ newly- born progenitor neurons to migrate to their destined target and became mature neurons.
[0087] These findings suggest that anti-EFRH treatment not only prevents cell death, followed by deterioration in cognitive functions, but also may promote neurogenesis and thus recovery from AD or other neurodegenerating diseases related to A/3P overproduction and neurotoxicity.
[0088] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
[0089] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
[0090] All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the
references cited within the references cited herein are also entirely incorporated by references.
[0091] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
[0092] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
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