WO2022140852A1 - Protéines stim couplant des récepteurs nmda à l'activation de pannexine 1 - Google Patents

Protéines stim couplant des récepteurs nmda à l'activation de pannexine 1 Download PDF

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WO2022140852A1
WO2022140852A1 PCT/CA2021/051892 CA2021051892W WO2022140852A1 WO 2022140852 A1 WO2022140852 A1 WO 2022140852A1 CA 2021051892 W CA2021051892 W CA 2021051892W WO 2022140852 A1 WO2022140852 A1 WO 2022140852A1
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panxl
stim
activation
panx1
amino acids
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Michael F. JACKSON
Chetan PATIL
Natalie LAVINE
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University Of Manitoba
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Glutamatergic signalling plays a critical role in diverse processes linked to learning and memory formation.
  • Ca 2+ signals generated by the NMDA subtype of glutamate receptors (NMDARs) play an indispensable role in long term potentiation (LTP), a process crucial in memory formation (1-3).
  • NMDAR signalling is also associated with increasing synaptic strength by initiating signalling events within the endoplasmic reticulum (ER) (4). Indeed, Ca 2+ entry via NMDARs is recognized to promote Ca 2+ induced Ca 2+ release (CICR) from ER stores through ryanodine (RyR) and IP3 receptor (IP3R) stimulation (5).
  • CICR Ca 2+ induced Ca 2+ release
  • RhyR ryanodine
  • IP3R IP3 receptor
  • STIMs stromal interaction molecules
  • CAG Ca 2+ release activated channels
  • Pannexinl (Panx1 ) channels can likewise be activated by STIM proteins.
  • Panxl is a large pore non-selective ion and solute permeable channel with prominent CNS expression (9-11). Panxl activation has been linked to pathophysiological disorders such as ischemia, stroke, migraine, chronic pain and epilepsy (12, 13). However, Panxl also mediates physiological processes in the CNS, including contributions to NMDAR-dependent synaptic plasticity underlying learning and memory (14), neural stem cell maintenance, spine formation and network connectivity (15, 16). In a manner analogous to NMDARs, the consequence of Panxl activation appears dichotomous due to its role in physiological and pathophysiological processes. But, how does NMDAR-Panxl differentiate between these opposite signals?
  • STIM proteins are single pass transmembrane proteins with Ca 2+ binding luminal EF hand and cytosolic protein-interacting domains (17). In eukaryotes, STIM are present in two isoforms- STIM1 and STIM2 with similar function and distribution, and are present as inactive dimers when Ca 2+ is bound to the EF hand. However, when the Ca 2+ stores are depleted, STIM proteins undergo conformational change followed by oligomerization. STIM oligomers translocate to ER-plasma membrane (PM) junctions where they interact with and activate PM channels (e.g. Orail ) through protein-protein interaction.
  • PM ER-plasma membrane
  • SOC store-operated or capacitative Ca 2+ entry
  • CCE capacitative Ca 2+ entry
  • STIM-operated signalling represents a general mechanism for coordinating intracellular and plasma membrane signalling events is supported by the identification of several STIM associated proteins, such as the calmodulin, the Ca 2+ regulated adaptor protein CRACR2 (Ca 2+ release-activated Ca 2+ receptor 2), and other STIM-regulated proteins including voltage-gated Ca 2+ channels, TRP channels, adenylyl cyclases and possible /?-channels (8, 22).
  • Our data adds to evidence supporting an important role of STIM proteins in relaying events engendered within the ER to PM channels and adds a new dimension to understanding Panxl functions linked to NMDAR initiated signalling events.
  • a method of treating a pathopsychological disorder associated with Panxl activation comprising administering an effective amount of a Panxl activation inhibiting agent to an individual in need of such treatment.
  • a method for screening for agents able to specifically prevent Panxl activation by STIM comprising: administering to a cell expressing Panxl an agent of interest, an amount of thapsigargin effective to activate Panxl and an effective amount of a Panxl - permeable dye; and quantifying entry of the Panxl -permeable dye into the cell; wherein if the quantity of the Panxl -permeable dye in the cell is below a control level quantified from a cell exposed to similar conditions as set forth above except for the presence of the agent of interest, the agent of interest is able to prevent Panxl activation.
  • ER calcium store depletion activates Panxl channels in hippocampal neurons.
  • a representative trace image for voltage-clamp recording in a cultured hippocampal neuron shows activation of a large inward current upon 3 pM thapsigargin (Tg) application. This current is blocked by 100 pM lanthanum (La 3+ ).
  • Tg 3 pM thapsigargin
  • La 3+ 100 pM lanthanum
  • the IV plot shows the Tg induced current with a linear current-voltage relationship and a reversal potential near 0 mV.
  • B1-B2 Similar observations are made upon application of cyclopiazonic acid (CPA).
  • C1 At the time indicated by the bar, isotonic NMDG-CI (140 mM) and CaCl2 solution (120 mM) were applied externally to the Tg-induced current. Both the treatments reduced 50% of the current previously carried by Na + .
  • C2 Summary data for average holding current at -60 mV.
  • D Summary of average holding current at -60mV recorded from Tg and CPA applied neurons.
  • E Tg induced Panxl currents are significantly reduced in neurons cultured from Panxl KO mice when compared to Panxl WT neurons.
  • F Application of Panxl inhibitors significantly reduced Tg induced Panxl currents in CD1 neurons. Data represented as mean ⁇ SEM.
  • Fig. 2 Calcium store depletion activates Panxl channels in a STIM dependent manner.
  • A-B Voltage-clamp recordings show ER Ca 2+ store depletion by thapsigargin (Tg, 3 pM) does not induce inward currents in non-transfected HEK 293T cells and HEK 293T cells transiently transfected with mPanxI alone.
  • C1 Tg induces large inward current in HEK 293T cells transiently co-transfected with mPanxI and mSTIM2. This current is blocked upon application of 100 pM lanthanum (La 3+ ).
  • Fig. 3 Illustration for N/C term deletion constructs of mPanxI . Schematic representation of Panxl channel membrane topology. Deletion of entire C-term of Panxl was termed as Panxl A299-426. When only distal C-term of Panxl was deleted from amino acid 378 (caspase cleavage site), this was termed as Panxl A379- 426. Deletion of entire N-term of Panxl was termed as Panxl A1 -35. Also, the N term of Panxl was deleted in two halves, Panxl A1 -18 (first 18 amino acids deleted) and Panxl A19-35 (next 17 amino acids deleted).
  • Panxl A1 -6 and Panxl A1 -12 consists of deletions of first 6, and 12 amino acids respectively.
  • Panx1A13-18 consists of deletion of amino acids from 13-18 only.
  • Panxl -STIM 1/2 interaction domain lies within first 18 amino acids of Panxl N-term.
  • WT mPanxI Full length mPanxI
  • Panxl A379-426 and every N term deletion variants except Panxl A19-35 shows localization similar to that of full length Panxl (WT Panxl ).
  • Panxl A19-35 is localized predominantly in the cytoplasm or trapped in the ER. Immunostaining was done on transfected HEK 293T cells using Flag antibody (Panxl constructs- green). Cell nuclei were stained using DAPI.
  • Panxl -STIM The interaction between Panxl -STIM was studied using co-immunoprecipitation.
  • HEK 293T cells were transfected with flag- tagged eGFP (negative control) or mPanxI variants with mSTIMI .
  • Co-precipitated proteins using anti-Flag beads were identified by western blot.
  • the blots were probed for STIMI and Panxl variants (using STIM1 and FlagM2-HRP antibody).
  • Panxl bound STIM protein was detected in protein fractions from WT Panxl (Flag-mPanx1 ) and Panxl A379-426.
  • loss of Panxl -STIM interaction was observed in cells transfected with Panx1A1 -18.
  • NMDAR stimulation activates downstream Panxl currents in hippocampal neurons.
  • A1-B Voltage-clamp recordings in a hippocampal neuron confirms development of La 3+ sensitive Panxl currents downstream of NMDAR stimulation (100 pM NMDA applied for 5 min). The NMDA evoked Panxl currents were blocked in presence of APV (100 pM, blocks glutamate binding in NMDARs) or MK-801 (20 pM, blocks channel pore and Ca 2+ influx through NMDARs) or 7-KYNA (10 pM, blocks glycine binding in NMDARs).
  • (A2) l-V curve analysis reveals development of large Panxl currents with linear l-V characteristics passing through 0 mV and blocked by La 3+ .
  • (B) Summary data for NMDAR-initiated Panxl currents in CD1 neurons (DIV 18-21 ). One-way ANOVA analysis revealed significant difference in Panxl currents when NMDAR antagonists treated groups were compared to that of control. **** p ⁇ 0.0001 , n 6-10 cells.
  • NMDAR-initiated Panxl currents are RyR/IP3R dependent in hippocampal neurons.
  • A In a representative neuron, a baseline recording period was followed by a continuous bath application of NMDA (100 pM) for 5 minutes. Sustained large Panxl currents were recorded after washout of NMDA and were inhibited by 100 pM lanthanum (La 3+ ). The amplitude of Panxl currents was suppressed in a neuron treated with 50 pM dantrolene (Dan, RyR/IP3R blocker).
  • B Summary data from a series of comparable recordings demonstrating that Panxl currents triggered by bath applied NMDA are suppressed in neurons pre-treated with Dan.
  • Fig. 7 NMDAR-initiated Panxl currents requires STIM1/2 in hippocampal neurons.
  • A Representative image confirming expression of mPanxI -eGFP and mCherry-mSTIM2 in lentivirus infected Panxl KO neurons assessed through fluorescence microscopy. Panxl is localized on the plasma membrane of neurons and STIM2 is distributed in a reticular manner within the ER.
  • B NMDA treated neurons (100 pM, 5 min) shows STIM puncta formation which represent STIM oligomerization and causes increased co-localization with mPanxI on the plasma membrane (marked with arrows).
  • C Cultured hippocampal neurons from CD1 mice (3-6 DIV) were infected with lentivirus containing shRNAscrambied, shRNAsTiMi-hpi or shRNAsTiM2-h Pi plasmids. Neurons were lysed 14-16 days after infection using standard procedures. Detergent-solubilized whole-cell protein extracts were analyzed by western blotting with antibodies recognizing STIM1 (C1), STIM2 (C2) and (3-actin (C1-2). Representative blots are shown. Results from 3 experiments were quantified densitometrically and band intensities were normalized to (3-actin.
  • Fig. 8 Functional importance of Panxl N-term.
  • A Summary data for voltageclamp recordings in hippocampal neurons cultured from Panxl WT/KO mice. NMDAR-initiated Panxl currents are significantly reduced in Panxl KO neurons when compared to Panxl WT neurons. The phenotype of Panxl KO neurons was rescued upon exogenous expression of mPanxI tagged to eGFP using lentiviral transfection. No difference was observed in NMDAR-initiated Panxl currents between Panxl WT and Panxl KO rescued neurons.
  • NMDAR-initiated Panxl currents were reduced in Panxl KO neurons expressing A1 -18 mPanxI when compared to Panxl WT and Panxl KO rescue (with mPanxI ) neurons.
  • the phenotype of Panxl KO neurons was not rescued upon exogenous expression of A1 -18 mPanxI .
  • No difference was observed in NMDAR-initiated Panxl currents between Panxl WT and Panxl KO A379-426 mPanxI rescued neurons.
  • FIG. 1 Representative image confirming expression and cell surface localization of mPanxI , A1 -18 mPanxI and A379-426 mPanxI in Panxl KO neurons assessed through fluorescence microscopy. The infected neurons were stained with mouse Anti-Flag primary antibody (mPanxI DNA constructs consists of Flag tag) and visualized with Anti-mouse Alexa 555.
  • C HEK 293T cells transfected with WT Panxl and A1 -18 Panxl show differential staining with Panxl monoclonal antibody from Cell Signaling (Anti-Panx1 (OS)). Anti-Panx1 (CS) fails to detect A1 -18 Panxl when compared to Panxl WT.
  • OS Anti-Panx1
  • Antibody targeting C-term (Anti-Panx1 CT395) readily detects A1 -18 Panxl .
  • the transfected HEK 293T cells were stained with rabbit Anti-Panx1 primary antibody (CS and CT395) and visualized with Anti-rabbit Alexa 488. Cell nucleus was stained using DAPI.
  • Fig. 9 Panxl -STIM1/2 interaction model. 1. During resting state, the ER Ca 2+ stores are full. STIM1/2 proteins are stable and present as dimers with Ca 2+ ions bound to its EF-hand/SAM domain. Panxl channels on the plasma membrane are closed. 2. When ER Ca 2+ stores are depleted, the STIM1/2 proteins become unstable and oligomerize. The STIM1/2 oligomers are stabilized by SOAR domains (STIM1 Orai Activating Region, amino acid region 344-442) and they translocate to the ER membrane junctions in close proximity to the plasma membrane.
  • SOAR domains STIM1 Orai Activating Region, amino acid region 344-442
  • the poly-Lys rich tails interact with the phospholipids in the plasma membrane and stabilize the STIM1/2 oligomers exposing SOAR domains to interact with the N term of Panxl channels.
  • STIM1/2 activates Panxl channels leading to Ca 2+ influx through Panxl channel pore and ER Ca 2+ store replenishment. 3.
  • the STIM1/2 oligomers rapidly dissociate, deactivate Panxl channels and return to their Ca 2+ bound dimer forms and the system returns to the resting state.
  • STIM Under conditions of stress (such as hypoxia or excitotoxicity), we propose STIM can keep Panxl in activated state for longer durations leading to cell death.
  • Amyloid beta oligomer (A[3O) treatment increases Panxl channel activation in response to NMDAR stimulation of cultured neurons.
  • A Western blot analysis demonstrating the presence of higher order amyloid beta oligomers in conditioned media (CM) from 7PA2 cells but not in CHO-CM. A[3Os are visualized using an antibody selective for amyloid-beta.
  • B Amyloid-beta (1 -X) can readily be detected by ELISA in conditioned media from 7PA2 but not from CHO cells.
  • C Cultured hippocampal neurons from CD1 mice were treated with 7PA2-derived A[3Os (21 .5 pM A
  • A Cultured hippocampal neurons from CD1 , and Panxl KO mice were treated with A[3Os (21 .5 pM A
  • 31 -42) for 72 hrs. Representative trace for miniature excitatory post synaptic currents (mEPSC) recorded from control and 72 hrs A[3Os treated neurons is shown. A[3O treatment reduced the frequency of the mEPSCs (B) in A[3Os treated CD1 and KO Rescue (expression of Panxl WT through lentivirus) compared to controls. Panxl KO neurons showed no significant difference. No difference was observed in the amplitude of the mEPSCs (C). Statistical analysis carried out using one-way ANOVA Bonferroni post hoc test, ** p ⁇ 0.01 , n 4-10 cells (B-C).
  • Fig. 12 Tat-Panx1 9 ’ 18 (SEQ ID NO:4) and Tat-Panx1 1 ’ 12 (SEQ ID NO:3) inhibits NMDAR-stimulated Panxl -mediated currents.
  • A Representative cartoon explaining the strategy to create 2 Tat-conjugated membrane permeable peptides covering the Panxl -STIM interaction region (1 -18 of Panxl N term).
  • TAT-Panx1 9-18 peptide mimics region from 9-18 whereas, TAT-Panx1 1-12 mimics region from 1 -12 of Panxl N term respectively. Additionally, a scrambled control peptide for TAT-Panx1 9-18scr was generated.
  • FIG. 1 Summary of voltage clamp recordings in hippocampal neurons cultured from CD1 mice.
  • the NMDAR-initiated Panxl activation is observed in control (untreated) cells.
  • neurons treated with cell permeable Tat-Panx1 9-18 at 10 pM for 24-40 hrs or with Tat-Panx1 1-12 at 5 pM for 6 hrs showed reduced Panxl activation downstream of NMDAR- stimulation.
  • Data represented as mean ⁇ SEM, ***p ⁇ 0.001 by one-way ANOVA with post hoc Bonferroni test, n 6-11 cells each.
  • C Staining of Tat-Panx1g-18 treated neurons with Anti-Panx1 (CS) reveals distribution in close association to STIM1 .
  • Both treated and untreated neurons were stained with rabbit Anti-Panx1 (CS) antibody (detects Tat-Panx1 9 ’ 18 ) and mouse anti-STIM1 ; visualized with Anti-rabbit Alexa 488 and Anti-mouse Alexa 555.
  • CS rabbit Anti-Panx1
  • Fig. 13 Strategy for developing Panxl -STIM interaction displacing peptide mimicking Panxl N term.
  • A Summary data for voltage-clamp recordings in hippocampal neurons cultured from Panxl WT/KO mice. NMDAR-initiated Panxl currents are significantly reduced in Panxl KO and KO+A1 -18 mPanxI neurons when compared to Panxl WT neurons. The phenotype of Panxl KO neurons was rescued upon exogenous expression of A13-18 mPanxI using lentiviral transfection. No difference was observed in NMDAR-initiated Panxl currents between Panxl WT and Panxl KO+A13-18 mPanxI rescued neurons.
  • N term tail of Panxl (first 36 amino acids) is conserved across multiple species (human, SEQ ID NO:9; mouse, SEQ ID NO: 14; Rat, SEQ ID NO: 15; Zebrafish, SEQ ID NO: 16; Orangutan, SEQ ID NO: 17; Chicken, SEQ ID NO: 18; Goat, SEQ ID NO: 19).
  • the first 18 amino acids (where Panxl -STIM interaction domain is present and underlined in the figure) form a highly conserved motif within the N term tail of Panxl .
  • FIG. 14 Anti-Panx1 (CS) antibody can bind to and detect Tat-Panx1 9-18 peptide treated cells.
  • HEK 293T cells were treated with membrane permeable Panxl peptides (5 pM each for 1 hour) and were stained using Anti-Panx1 (CS) antibody to study peptide uptake. The antibody specifically detected only TAT-Panx1 9-18 peptide.
  • the treated HEK 293T cells were stained with rabbit Anti-Panx1 primary antibody (CS) and visualized with Anti-rabbit Alexa 488. Cell nucleus was stained using DAPI.
  • Panxl As described herein, we identified Panxl as a new binding partner of STIM proteins. Moreover, we demonstrate how ER Ca 2+ store depletion induces activation of Panxl channels in a STIM dependent manner. Using Panxl deletion variants, we identified the STIM interaction domain within the intracellular N-terminus of Panxl (amino acids 1 -18), which is necessary for promoting Panxl activation.
  • the 18-mer sequence is as follows: MAIA(H/Q)LATEYVFSDFLLK (SEQ ID No:1 ). The emerging view is that H is more prevalent in humans (as in other species) while substitution of Q to H at 5th amino acid position has no clinical effect (PMID- 28142297).
  • Panxl activation and increased ATP release Given the expression and critical function of Panxl channels, current research is focused on establishing mechanisms leading to Panxl activation and increased ATP release. Highlighting its responsiveness to multiple cellular and micro- environmental cues, till date numerous Panxl activation modalities have been reported. These include activation by elevated extracellular potassium (33, 34), caspase cleavage of the Panxl C-terminal tail (35, 36), tyrosine phosphorylation by Src family kinases (SFKs) (37, 38), mechanical stimulus (39, 40), and increased intracellular calcium (41). Despite aforementioned mechanisms, the interplay between the physiological and pathophysiological role of Panxl channels remains poorly understood.
  • Panxl channels In this study, we identified activation of Panxl channels as a consequence of ER Ca 2+ store depletion and further characterized this Panxl activation as STIM dependent.
  • Past studies have reported store operated Ca 2+ entry through physical protein-protein interaction between STIM and Orail channels (6, 7); however our work marks the introduction of Panxl channels as a new binding partner for STIM and a candidate channel for replenishing the ER Ca 2+ stores.
  • the association of Panxl -STIM can play a significant role in unidentified physiological as well as pathophysiological functions.
  • the channel In this state, the channel possesses a smaller conductance and on the basis of which has been dubbed the small pore state.
  • Panxl In its large pore state, Panxl has a larger conductance, linear IV with reversal at 0 mV (reflecting its non-selective ion permeability) and readily be blocked by La 3+ , but less readily by CBX.
  • HEK 293T expressing Panxl we note that coexpression of STIM proteins did not alter small pore properties of Panxl as outward rectification, reversal potential and peak current amplitudes were identical with or without STIM expression.
  • the channel demonstrates linear IV with reversal potential close to 0 mV. This shift in reversal potential is suggestive of the transition between small pore and large pore of the channel.
  • the domain of interaction between STIM and Orail is well studied and critical amino acid residues responsible for protein-protein interaction have been identified (7).
  • the C-term of STIM1 consists of a coiled-coil domain wherein the STIM-Orai activating region (SOAR) is present.
  • SOAR domain is -100 amino acids long and helps in the coupling of STIM1 with Orail channels (43).
  • Orail channels the short coiled-coil domain located in the intracellular C-terminus has been shown to play an important role in interaction with STIM1 (6).
  • a cluster of acidic residues (amino acids from 272-291 ) were identified as necessary for STIM1 binding and channel activation (7).
  • the 13-18 region (SDFLLK, SEQ ID No:2) is the minimum region necessary to allow binding and activation by STIM.
  • AD Alzheimer’s disease
  • AD Alzheimer’s disease
  • Panxl activation and 3) that treatment of neurons with a fusion peptide comprising a membrane permeable peptide, such as, for example, HIV-TAT, although other suitable membrane permeable peptides will be readily apparent to one of skill in the art and are within the scope of the invention and a Panxl peptide comprising or consisting or consisting essentially of an amino acid sequence as set forth in any one of SEQ ID Nos: 1 -4, for example, amino acids 1 -36 (SEQ ID NO: 9), 7-24 (SEQ ID NO: 10), 1 -18 (SEQ ID No:1 ), 1 -12 (SEQ ID No:3), 13-18 (SEQ ID No:2) or 9-18 (SEQ ID No: 4) of Panxl can likewise prevent Panxl activation by NMDARs by acting as a competitive antagonist able to prevent STIM-Panx1 interaction.
  • a fusion peptide comprising a membrane permeable peptide, such as, for example, HIV-TAT,
  • the displacing peptide may “consist essentially of” the recited peptides in that in some embodiments the peptide may comprise additional amino acids which may or may not be native to the Panxl sequence but which will not alter the ability of the recited peptide to prevent STIM association, that is, these additional, non-essential amino acids will not change the or alter or diminish the ability of the recited peptides to prevent Panxl activation.
  • the full length Panxl protein is 426 amino acids in total and the region 1 -36 represents the intracellular N-terminus.
  • the full length Panxl protein is largely present at the plasma membrane, with no soluble cytosolic fragments (i.e. N-term) normally expressed. Accordingly, the development of peptides able to bind STIM and thus prevent binding/activation of Panxl is very different from the native protein.
  • amino acids 19-36 (SEQ ID NO: 13), for example, amino acids 25-36 (SEQ ID NO: 11), represents a region that mediates distinct channel functions; and 2) that a selective reagent can be developed to alter functions mediated via this region.
  • some Panxl gain of function mutations have been reported and a treatment able to target Panxl (19-35, SEQ ID No:5) that reduce surface expression of Panxl would be valuable, for example, for conditions in which Panxl activity is increased (e.g. in AD).
  • the STIM interaction region of Panx1 is at amino acids 13-18 (SEQ ID NO: 2). This region is required to stimulate the pathological activation of Panxl , a response linked to detrimental actions of amyloid beta.
  • This technology provides a target to treat Alzheimer’s disease, as discussed above. This make take the form of membrane permeable STIM-displacing peptides (TAT-conjugated), STIM-displacing antibodies, small molecules, or any other method of blocking the specific interaction.
  • a method of treating a pathopsychological disorder associated with Panxl activation comprising administering an effective amount of a Panxl activation inhibiting agent to an individual in need of such treatment.
  • the pathopsychological disorder is a pathological consequence of NMDAR over activation.
  • the pathopsychological disorder may be selected from the group consisting of ischemia, stroke, migraine, chronic pain, epilepsy and Alzheimer’s disease.
  • the Panxl activation inhibiting agent prevents impaired neuronal function by A
  • the Panxl activation inhibiting agent is a Panxl -STIM interaction displacing peptide.
  • the Panxl -STIM displacing peptide mimics the Panxl N terminus and/or prevents STIM-Panx1 interaction.
  • the Panxl -STIM displacing peptide is selected from a peptide comprising the amino acid sequence as set forth in any one of SEQ ID No: 1 - 4 (1 -18 MAIA(H/Q)LATEYVFSDFLLK, SEQ ID No:1 ; 13-18 SDFLLK, SEQ ID No:2; 1 - 12 MAIA(H/Q)LATEYVF SEQ ID NO:3; and 9-18 EYVFSDFLLK SEQ ID NO:4), or amino acids 1 -24 (SEQ ID NO: 12) or 7-24 (SEQ ID NO: 10) of Panxl .
  • the Panxl -STIM displacing peptide may further comprise a membranepenetrating domain.
  • the Panxl activation inhibiting agent is an antibody binding to N-terminus of Panxl .
  • the antibody binding to the N-terminus of Panxl may prevent STIM-Panx1 interaction.
  • the antibody binding to the N-terminus of Panxl may bind an epitope within amino acids 1 -36 (SEQ ID NO: 9) of Panxl , within amino acids 7-24 (SEQ ID NO: 10) of Panxl , within amino acids 25-36 (SEQ ID NO: 11 ) of Panxl , within amino acids 1 - 18 (SEQ ID NO: 1 ) of Panxl , within amino acids 19-35 (SEQ ID NO: 5) of Panxl , within amino acids 9-18 (SEQ ID NO: 4) of Panxl and/or within amino acids 13-18 (SEQ ID NO: 2) of Panxl .
  • the antibody binding to the N-terminus of Panxl is rabbit Anti-Panx1 primary antibody (Cell Signaling Technology (GST), product #91137S) and may bind to an epitope within amino acids 7-24 (SEQ ID NO: 10) of Panxl . While not wishing to be bound to a particular theory or hypothesis, it is believed that the anti-Panx1 (GST) antibody acts to prevent STIM binding and activation, but does not affect surface expression.
  • GST Cell Signaling Technology
  • an antibody binding to the epitope within amino acids 19-36 (SEQ ID NO: 13) of Panxl for example, amino acids 25-36 (SEQ ID NO: 11 ).
  • an antibody binds to the region of Panxl responsible for surface expression, which is restricted to amino acids 25-36 (SEQ ID NO: 11 ) and corresponds to a previously unidentified leucine rich region. While not wishing to be bound to a particular theory or hypothesis, it is believed that this antibody would have a separate mechanism of action, whereby it could reduce surface expression, without interfering with STIM binding.
  • the antibody binding to the N-terminus of Panxl has been modified for cell penetration.
  • the Panxl activation inhibiting agent may be an agent able to specifically prevent Panxl activation by STIM, for example, a small molecule.
  • a method for screening for agents able to specifically prevent Panxl activation by STIM comprising: administering to a cell expressing Panxl an agent of interest, an amount of thapsigargin effective to activate Panxl and an effective amount of a Panxl - permeable dye; and quantifying entry of the Panxl -permeable dye into the cell; wherein if the quantity of the Panxl -permeable dye in the cell is below a control level quantified from a cell exposed to similar conditions as set forth above except for the presence of the agent of interest, the agent of interest is able to prevent Panxl activation.
  • EXAMPLE 1 - ER store depletion activates Panxl in hippocampal neurons.
  • STIM proteins In non-neuronal cells, the process of store-operated calcium entry (SOCE) is carried out predominantly by STIM proteins and Orail channels.
  • STIM proteins expressed in neurons are increasingly implicated in physiological and pathophysiological processes (23), knowledge regarding the extent to which Orai channels are expressed in neurons and participate as STIM signalling partners is more limited.
  • STIM proteins have been shown to interact and regulate the activity of a variety of surface expressed channels, implying that STIMs subserve a broader range of cellular functions, in addition to their canonical role in SOCE.
  • Panxl is STIM-responsive, we stimulated cultured neurons with thapsigargin, a SERCA inhibitor known to recruit STIM.
  • thapsigargin (3 pM) reliably evoked slowly developing, large inward currents (Fig. 1 A) with linear current-voltage and reversal near 0 mV (Fig. 1 A, D), suggesting activation of a non-selective ion conductance.
  • the properties of thapsigargin-stimulated currents including spontaneously fluctuating peak amplitudes, linear IV and sensitivity to La 3+ , are consistent with Panxl channel activation.
  • Panxl is further supported by ion substitution experiments demonstrating that thapsigargin-stimulated currents are permeable to Ca 2+ as well as the large organic cation NMDG (Fig.
  • EXAMPLE 2 - Panxl channels are activated in a STIM dependent manner.
  • Panxl channels are responsive to thapsigargin, we sought to determine whether such activation is contingent on STIM.
  • STIM HEK 293T
  • Panxl can reside in a small pore open state with outwardly rectifying currents primarily carried by Cl’ and readily blocked by CBX (10). Small pore, outwardly rectifying currents can readily be detected in unstimulated cells that express Panxl .
  • Panxl and STIM transcripts can be detected endogenously in HEK cells, protein expression is absent for Panxl and modest for STIM1/2. Moreover, CBX-sensitive outwardly rectifying currents are absent in native HEK cells and thapsigargin application does not induce any membrane current (Fig. 2A, D). In contrast, outwardly rectifying CBX-sensitive currents could readily be recorded from HEK cells expressing Panxl . However, expression of Panxl alone was insufficient to reconstitute thapsigargin treatment-induced currents (Fig. 2B, D). Only when Panxl was co-expressed with either STIM1 or STIM2, did thapsigargin application evoke currents comparable to those recorded in neurons (Fig. 2C1 ).
  • Thapsigargin-stimulated currents exhibited linear IV characteristics (Fig. 2C2) and could be completely inhibited by application of La 3+ (Fig. 2C1 ,2).
  • the onset of thapsigargin-stimulated large inward currents was associated with a pronounced depolarizing shift in current reversal from ⁇ -60 mV to ⁇ 0 mV (Fig. 2C3), which is consistent with Panxl opening from its small pore, primarily Cl- permeable, to its large pore non-selective ion channel configuration.
  • STIM has been shown to interact with target channel constitutively (25, 26), irrespective of ER Ca 2+ store fill status, or inducibly, in response to store depletion.
  • IP immunoprecipitation
  • Panxl mutants in which most of the N- or C-term was deleted (Fig. 3).
  • Panxl A300-426) causes intracellular retention of Panxl (27)
  • caspase cleavage site mutant Panxl A379- 426) (28) and examined whether this variant is responsive to thapsigargin (Fig. 4A, B and D).
  • this mutant expresses well at the PM and generates robust CBX-sensitive outwardly-rectifying currents with negative reversal potential ( ⁇ - 60mV).
  • Panxl A379-426 When co-expressed with STIM, Panxl A379-426 was activated upon thapsigargin application, with no difference in IV characteristics or amplitude when compared to full length Panxl (Fig. 4B, D). This suggests that the STIM interacting domain does not reside within the distal C-term of Panxl . We therefore examined the role of the Panxl N-term.
  • Panxl A1 -18 was extensively localized at the plasma membrane, where it co-distributed with cortical F-actin, Panxl A19-35 was predominantly retained intracellularly, within the ER as evidenced by its co-distribution with calnexin (Fig. 4 and fig. S3B-C).
  • calnexin Fig. 4 and fig. S3B-C
  • thapsigargin stimulation does not provide insight regarding physiological mechanisms by which Panxl is activated in response to ER-initiated signalling events.
  • To identify a more physiological activation context we considered the well-known coupling between NMDAR activation and Ca 2+ induced Ca 2+ release from ER stores (5, 29).
  • NMDAR activation can recruit STIM (30), and consequently regulate the activity of voltage-gated Ca 2+ channels as well as AMPARs (8).
  • NMDAR stimulation can activate Panxl - mediated inward currents in a graded manner, contingent on the duration of NMDAR stimulation (31).
  • Panxl currents induced in response to NMDA treatment were prevented in neurons with NMDARs inhibited by structurally dissimilar agents that prevent glutamate (APV) or glycine (7-KYNA) binding, as well as by MK- 801 , a NMDAR pore blocker (Fig. 5B). Further, application of NMDA in extracellular solution devoid of added Ca 2+ , failed to evoke Panxl -mediated currents. Together, this data supports that the NMDAR-initiated currents are Panxl in nature and Ca 2+ influx through NMDARs is necessary for Panxl activation.
  • NMDAR stimulation is the activation of signaling cascades that lead to the activation of calcium receptors present on the ER (32). This has been shown to promote efflux of Ca 2+ from ER into the cytosol thereby partially depleting ER Ca 2+ stores and in turn activating STIM proteins. Consistent with this, block of Ryr and IP3Rs completely abolished Panxl activation in response to NMDA treatment (Fig. 6A-C).
  • mice Primary cultures of mouse hippocampal neurons were prepared as follows. Hippocampi were dissected from embryonic day 17-18 CD1 , Panxl WT or Panxl KO mice under sterile conditions. Dissected hippocampi were minced and placed in 0.25% trypsin-EDTA before mechanical dissociation by trituration.
  • HEK 293T cells were cultured and maintained using Dulbecco’s Modified Eagle Medium containing glutamine (DMEM, Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) in 100 mm cell culture dishes.
  • Dulbecco Modified Eagle Medium containing glutamine
  • FBS fetal bovine serum
  • HEK 293T cells were transfected using jetPRIME reagent (Polypus transfection, France) according to manufacturer’s guidelines.
  • For imaging cells were transfected with a Flag-tagged Panxl construct, with or without STIM1 or STIM2.
  • a plasmid expressing EGFP (47) was added to the Panxl /STIM transfection mix, which acted as a fluorescent marker for transfection efficiency and as a target for cell selection.
  • 24 hours post transfection the cells were re-seeded onto 35 mm culture dishes (for electrophysiology or biochemistry) or onto poly-D-lysine coated imaging grade plastic bottom dishes (Ibidi, Wl). Dishes were used within 24 hours of re-seeding.
  • For biochemistry cells were incubated for 48 hours post transfection and then lysed for western blot and immunoprecipitation assays.
  • Western Blotting Western Blotting
  • Transfected cells were washed with ECF, scraped and pelleted.
  • the pellet was resuspended in ComplexioLyte No. 48 lysis buffer (CL-48-LB; LogoPharm, Germany) and mechanically lysed by passing through a 25G needle 8-10 times.
  • the lysate was placed on a nutator mixer at 4°C for 30 min, followed by centrifugation at 21 ,000xg at 4°C for 10 min.
  • the supernatant contained the soluble fraction to be used for immunoprecipitation (IP). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo).
  • Sequences were based on the RNAi Consortium analysis using mouse sequences for STIM1 and STIM2. Each shRNA was screened in HEK 293T cells expressing either STIM1 or STIM2. Cells were lysed and equal amounts of protein loaded onto SDS-PAGE and separated. Using western blotting we identified the shRNA for each protein that was knocked down by >95%, which we are calling STIM1 -hp1 (5’-gcagtactacaacatcaagaa- 3’, SEQ ID No:6) and STIM2-hp1 (5’-cctctgtcataatggtgagaa-3’, SEQ ID No:7).
  • Lentiviral particles were prepared for pLB(EGFP)-STIM1 -hp1 and pLB(mCherry)-STIM2-hp1 by following a modified version of Trono Lab protocol (48). Briefly, HEK 293T cells were transfected with packaging vectors pSL3, pSL4, and pSL5, as well as the transfer plasmid (pLB construct), using lipofectamine 2000 (Invitrogen). Cells were allowed to incubate for 24-30 hours after which the media was collected into an Amicon Ultra-15ml 100k MWCO centrifugal filter device and centrifuged in a swinging bucket at 4,000xg for 15 minutes at 4°C to concentrate the viral particles.
  • the lentiviruses were used to transduce CD1 neurons in culture at DIV 3-6 and lysed at DIV 19 using RIPA buffer. Testing for knockdown was done by Western blotting. Constructs mRNA was obtained from mouse brain tissue using a kit (Qiagen). This was followed by RT-PCR using oligo(dT) as primers to produce a mixture of mouse brain cDNA, which was used as a template for cloning.
  • Oligos spanning the mouse genes of interest of Panxl (Accession #NM_019482.2), STIM1 (Accession #NM_009287.4) and STIM2 (Accession #NM_001081103.2 using the upstream AUG codon with a putative N-terminal extension) were designed, with restriction sites on either side for insertion into pcDNA3.1 (+).
  • flag tag (DYKDDDDK, SEQ ID No:8) was inserted onto the N-terminus of Panxl , EGFP tag was fused to the C-terminus of Panxl , and mCherry was inserted between the signal sequence for STIM1 (amino acid 1 to 21 ) or STIM2 (amino acid 1 - 86) and the rest of the protein.
  • Panxl deletion PCR constructs were as follows (with deleted amino acids preceded by a A): flag-Panx1 (A379-426), flag-Panx1 (A1 -18), flag- Panxl (A19-35), flag-Panx1 (A1 -6), flag-Panx1 (A1 -12), flag-Panx1 (A13-18).
  • Electrodes had a final resistance of 3-5 MQ when filled with intracellular fluid (IGF) containing (in mm): 142 cesium gluconate, 10 HEPES, 2 MgCL, 8 NaCI, pH 7.2 (adjusted with 1 M Cs-OH) and osmolarity between 290-295 mOsm/liter.
  • Standard extracellular fluid (ECF) was composed of (in mm): 140 NaCI, 5.4 KCI, 25 HEPES, 33 glucose, 2 CaCI 2 , 1 MgCL, 0.1 pm TTX, pH of 7.4 (adjusted with 10N NaOH) and osmolarity between 300-305 mOsm/liter.
  • TTX was only added to the ECF when recording from neurons.
  • HEK 293T cells cultured on ibidi dishes ibidi, Wl
  • neurons cultured on glass bottom dishes MatTek
  • PLP fixative 50
  • the dishes were washed with ECS thrice, followed by incubation in PLP fixative for 10 minutes at RT. After incubation the dishes were washed thrice for 5 mins using PBS. The fixed cells were then permeabilized using 0.2% Triton-X in PBS (for 10 mins).
  • the cells were blocked for 30 mins in PBS containing 5% normal serum based on the host of the secondary antibody used. After blocking, the cells were incubated with primary antibodies in 0.5% BSA in PBS for duration of 2 hours followed by three washes of PBS for 5 mins each. Cells were then incubated with fluorophore conjugated secondary antibodies in 0.5% BSA in PBS for 1 hour in the dark, followed by three washes of PBS for 5 mins each. The cells were additionally incubated with DAPI for 5 mins to stain the nuclei and washed with PBS thrice before preserving the dishes at 4 °C in PBS. The cells were imaged within two weeks of immunostaining.
  • pannexin 1 reveals unique motifs for ion selection and inhibition, eL/fe 9 (2020), doi:10.7554/eLife.54670.

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Abstract

Nous avons identifié Panx1 en tant que nouveau partenaire de liaison de protéines STIM et démontré comment l'appauvrissement en ER Ca2+ induit une activation de canaux Panx1 d'une manière dépendant du STIM. À l'aide de variants de délétion de Panx1, nous avons identifié le domaine d'interaction STIM à l'intérieur de l'extrémité N-terminale intracellulaire de Panx1 (acides aminés 1-18), qui est nécessaire pour favoriser l'activation de Panx1. En outre, dans les neurones de culture hippocampiques, nous avons démontré que la fonction ionotrope de NMDAR provoque l'activation de Panx1 et l'inactivation de STIM inhibe l'activation de Panx1. La région Panx1-STIM contrôle l'activation de Panx1 initiée par NMDAR, confirmant le modèle de signalisation NMDAR-STIM-Panx1. L'extrémité N-terminale (acides aminés 19-35) est responsable du trafic de la membrane plasmique des canaux Panx1. La région d'interaction STIM de Panx1 est au niveau des acides aminés 13-18. Cette région est nécessaire pour stimuler l'activation pathologique de Panx1, une réponse associée à des actions préjudiciables de l'amyloïde bêta. Cette technologie fournit une cible pour traiter la maladie d'Alzheimer.
PCT/CA2021/051892 2020-12-29 2021-12-28 Protéines stim couplant des récepteurs nmda à l'activation de pannexine 1 WO2022140852A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FLORES-MUÑOZ CAROLINA, GÓMEZ BÁRBARA, MERY ELENA, MUJICA PAULA, GAJARDO IVANA, CÓRDOVA CLAUDIO, LOPEZ-ESPÍNDOLA DANIELA, DURÁN-ANI: "Acute Pannexin 1 Blockade Mitigates Early Synaptic Plasticity Defects in a Mouse Model of Alzheimer’s Disease", FRONTIERS IN CELLULAR NEUROSCIENCE, vol. 14, 19 March 2020 (2020-03-19), pages 46, XP055955191, DOI: 10.3389/fncel.2020.00046 *
YEUNG ALBERT K., PATIL CHETAN S., JACKSON MICHAEL F.: "Pannexin‐1 in the CNS: Emerging concepts in health and disease", JOURNAL OF NEUROCHEMISTRY, vol. 154, no. 5, 1 September 2020 (2020-09-01), GB , pages 468 - 485, XP055955195, ISSN: 0022-3042, DOI: 10.1111/jnc.15004 *

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