WO2020081862A1 - Methods and compositions for modulating secretion of complement component 4 - Google Patents

Abstract

Disclosed herein are methods of modulating the secretion of complement component 4 (C4). Also disclosed herein are methods of treating a neurodegenerative or neuropsychiatric disorder by modulating secretion of C4.

Description

METHODS AND COMPOSITIONS FOR MODULATING SECRETION OF COMPLEMENT COMPONENT 4

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 62/747,110, filed on October 17, 2018, and U.S. Provisional Application No.

62/867,064, filed on June 26, 2019, the contents of which are hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Recent research on mouse astrocytes has highlighted their central role in the normal development and function of the central nervous system, as well as their potential participation in many pathological conditions (reviewed in Sofroniew MV, Vinters HV., 2010 and Tyzack G., 2016). Astrocytes are crucial for the formation and remodeling of synapses, and recent literature has proposed that the complement component and synaptic pruning are hallmarks of schizophrenia. However, the effect of the complement component on other neurodegenerative and/or neuropsychiatric diseases will benefit from further elucidation.

SUMMARY OF THE INVENTION

[0003] In some aspects, the disclosure provides methods of treating a neurodegenerative and/or neuropsychiatric disorder comprising administering an agent that modulates secretion of complement component 4 (C4), thereby treating a neurodegenerative and/or neuropsychiatric disorder.

[0004] In some embodiments, the agent downregulates secretion of C4. In some embodiments, the agent targets one or more pathways involved in C4 secretion. In some embodiments the pathway is a pathway shown in Table 1. In some embodiments, the neurodegenerative and/or neuropsychiatric disorder is selected from the group consisting of Alzheimer’s disease, Rett Syndrome, Huntington Disease, Multiple Sclerosis, and Schizophrenia. In some embodiments the agent is an agent identified in Table 1.

[0005] In some embodiments, the agent targets BET. For example, the agent may be a BET bromodomain inhibitor (e.g., an inhibitor of one or more of BRD2, BRD3, and BRD4). In some embodiments, the agent targets JAK. For example, the agent may be a JAK inhibitor (e.g., an inhibitor of one or more of JAK1, JAK2, and JAK3). In some embodiments, the agent targets Akt. For example, the agent may be an Akt inhibitor (e.g., an inhibitor of one or more of Aktl, Akt2, and Akt3). In some embodiments, the agent targets p38 MAPK. For example, the agent may be a p38 inhibitor (e.g, an inhibitor of one or more of p38a and r38b). In some embodiments, the agent targets histone deacetylase (HD AC). For example, the agent may be an HDAC inhibitor (e.g., an HDAC6 inhibitor). In some embodiments, the agent targets DNA methyltransferase (DNMT). For example, the agent may be a DNMT inhibitor (e.g, an inhibitor of one or more of DNMT1, DNMT3A, and DNMT3B). In some embodiments, the agent targets phospholipase A (PLA). For example, the agent may be a PLA inhibitor (e.g., a non-pancreatic secretory phospholipase A2 inhibitor). In some embodiments, the agent targets ferroptosis. For example, the agent may be a ferroptosis activator. In some embodiments, the agent targets Src. For example, the agent may be a Src inhibitor (e.g, a dual Src/Abl inhibitor). In some embodiments, the agent targets Bruton’s tyrosine kinase (BTK). For example, the agent may be a Btk inhibitor. In some embodiments, the agent targets BMI. For example, the agent may be a BMI inhibitor (e.g., a BMI-l inhibitor). In some embodiments, the agent targets PARP. For example, the agent may be a PARP inhibitor (e.g., a PARP1 inhibitor). In some embodiments, the agent targets S1P receptor. For example, the agent may be a S1P antagonist. In some embodiments, the agent targets IkB/IKKI. For example, the agent may be an inhibitor of IKK (e.g., an IKKb inhibitor). In some embodiments, the agent targets DPP-4. For example, the agent may be a DPP-4 inhibitor. In some embodiments, the agent targets IGF-1R. For example, the agent may be an IGF-1R inhibitor. In some embodiments, the agent targets p97. For example, the agent may be a p97 inhibitor. In some embodiments, the agent targets Bel -2. For example, the agent may be a Bcl-2 inhibitor. In some embodiments, the agent targets Chk. For example, the agent may be a Chk inhibitor (e.g., an inhibitor of one or more of Chkl and Chk2). In some embodiments, the agent targets PPAR. For example, the agent may be an antagonist of PPAR (e.g., a PPARy antagonist).

[0006] In some embodiments, the agent targets an epigenetic pathway. In some embodiments, the agent targets a JAK/STAT pathway. In some embodiments, the agent targets a PI3K/Akt/mTor pathway. In some embodiments, the agent targets a MAPK pathway. In some embodiments, the agent targets a metabolism pathway.

In some embodiments, the agent targets an angiogenesis pathway. In some embodiments, the agent targets a GPCR and/or G protein pathway. In some embodiments, the agent targets aNF-kB pathway. In some embodiments, the agent targets a proteases pathway. In some embodiments, the agent targets a protein tyrosine kinase pathway. In some embodiments, the agent targets an ubiquitin pathway. In some embodiments, the agent targets an apoptosis pathway. In some embodiments, the agent targets a cell cycle pathway. In some embodiments, the agent targets a DNA damage pathway. In some embodiments, the agent targets a transmembrane transporter pathway. In some embodiments, the agent targets an endocrinology and hormone pathway. In some embodiments, the agent targets a neuronal signaling pathway. In some embodiments, the agent targets a TGF- beta/SMAD pathway. In some embodiments, the agent targets a microbiology pathway. In some embodiments, the agent targets a cytoskeletal signaling pathway.

[0007] In some embodiments, the agent is IMD0354. In some embodiments, the agent is Tofacitinib. In some embodiments, the agent is selected from Table 1.

[0008] In some embodiments, the neurodegenerative and/or neuropsychiatric disorder is schizophrenia. In some embodiments, the neurodegenerative and/or neuropsychiatric disorder is Alzheimer’s Disease. In some embodiments, the modulation of C4 secretion results in the rescue of synaptic over-pruning.

[0009] In some aspects, the disclosure provides methods of modulating secretion of complement component 4 (C4) comprising administering an agent, wherein the agent is a downregulator of C4 secretion.

[0010] In some aspects, the disclosure provides methods of modulating secretion of complement component 4 (C4) comprising administering an agent, wherein the agent targets one or more pathways involved in C4 secretion. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0012] FIGS. 1A-1G demonstrate high-throughput small molecule screening to identify modulator of complement component 4 (C4). FIG. 1A provides a schematic of the screening timeline. FIG. 1B provides a bar graph of pathways involved in the regulation of C4. FIG. 1C provides a bar graph showing the targets of the identified compounds. FIG. 1D shows the relationship between the INFy pathway and JAK inhibitors. FIGS. 1E-1G provide dose-response curves of selected compounds.

[0013] FIGS. 2A-2D demonstrate primary screening analysis, secondary screening validation and dose response in stem-cell derived astrocytes. FIG. 2A provides a representative scatter plot showing the effect of compounds on C4 secretion (black squares represent average of triplicates) compared to DMSO (red squares, average of triplicates) at 1 mM. FIG. 2B provides a representative graph of the pipe line used for nuclei selection. Nuclei were counted using Columbus. Red square represent DMSO controls in the triplicates plates, Black squares represents triplicates. Nuclei below 3 SD of the average of DMSO control were excluded from further analysis. FIG. 2C shows secondary screening performed in 4 different concentrations per compound (3, 1, 0.3, 0.1 mM) in triplicates. Graph show the average of triplicates relative to DMSO (1). FIG. 2D shows dose-response curve of two selected compounds (+)- JQ1 and FTY720 on stem cell-derived astrocytes (Mito 23 Schizoaffected; Mito 80 Bipolar). Cell lines C4 copy number notes: Mito 234 - C4A(3), C4B(l), C4L(4), C4S(0), total CNV(4); Mito 80 - C4A(3); C4B(l); C4L(3); C4S(l); total CNV(4).

[0014] FIG. 3 provides a schematic of how BET inhibitors work. BET- proteins interact with Ac Lysine on histone tails to promote active transcription (left panel). BET inhibitors (e.g., (+)- JQ1) preclude the interaction of BET-proteins with Ac Lysine (right panel).

[0015] FIGS. 4A-4C demonstrate (+)- JQ1 represses transcription of C4 and interferes with pro-inflammatory signals. FIG. 4A shows BRD4 displacement from chromatin in astrocytes treated with (+) - JQ1 luM for 24 hours compared to DMSO control. FIG. 4B shows qPCR for the expression of C4A and C4B in biological triplicates of stem cell-derived astrocytes (HA) treated with DMSO or JQ1 for 24 hs. FIG. 4C shows secretion of C4 measured by ELISA of stem cell-derived astrocytes treated with DMSO, JQ1 (luM), pro-inflammatory stimuli INFy (250 ng/mL) alone, INFy in combination with JQ1, Polyinosinic:polycytidylic acid poly I:C (10 mg/mL), or poly I:C in combination with JQ1. Data are represented as mean ± SD relative to DMSO control (100% secretion) using unpaired t-test **** p < 0.0001.

[0016] FIGS. 5A-5D demonstrate (+)- JQ1 regulates transcription of other complement components and cytokine secretion. FIG. 5A provides a bar graph quantifying the amount of BRD4 present on the chromatin fraction upon treatment with DMSO or JQ1. FIG. 5B shows qPCR of the expression of different complement components in biological triplicates of stem cell-derived astrocytes treated with JQ1. FIG. 5C shows human cytokine array quantification of secretion comparing untreated stem cell-derived astrocytes with treatment with JQ1 for 24 hours. Data are represented as technical duplicates. FIG. 5D shows JQ1 treatment decreases complement components mRNA expression.

[0017] FIGS. 6A-6F demonstrate in vitro engulfment assay. FIG. 6A shows co-culture of Ngn2 neurons and astrocytes. FIG. 6B shows synptosomes purification (SynPER). FIG. 6C shows labelling of synaptosomes (pHrodo). FIG. 6D shows live imaging of engulfment. FIGS. 6E-6F provide quantification of engulfment with live imaging (FIG. 6E) and FACS analysis (FIG. 6F).

[0018] FIGS. 7A-7E demonstrate in vivo validation of JQ1. FIG. 7A provides a timeline of in vivo validation of JQ1 in a mouse. FIGS. 7B-7D show C4 expression in retina (FIG. 7B), frontal cortex (FIG. 7C), thalamus (FIG. 7D), and spleen (FIG. 7E) of mice treated with DMSO and JQ1 (5 mg/kg).

[0019] FIGS. 8A-8B demonstrate checking effect of JQ1 on the expression of C4 by ddPCR in C4 humanized mice using in vivo synaptic pruning assay. FIG. 8A provides a timeline of an in vivo synaptic pruning assay. FIG. 8B shows

retinogeniculate projection patterns visualized after injecting b-cholera toxin conjugated to Alexa 594 (€Tb-594) dye (red) and OTb^dd^Gbbh) into left and right eyes ofWT and Clq KO mice. See Stevens B. et al., Cell (2007) 131(6): 1164-1178.

[0020] FIG. 9 provides a schematic of the interactions of Fingolimod (FTY720) in a cell. The effect of FTY720 on astrocytes include inhibition of pro- inflammatory cytokine production, stimulation of cells migration, inhibition of astrogliosis, and downregulation of NFKB pathway signaling.

[0021] FIGS. 10 A- 10C demonstrate treatment with Fingolimod (FTY720) activates the pro-survival signaling. FIG. 10A provides blot showing results of treatment with FTY720 (1 mM) for 15 min, 30 min, and 24 hours. FIG. 10B shows immunocytochemistry of pERK in cells treated with DMSO and FTY720. Blue, dapi staining. FIG. 10C shows percent of pERK nuclei of cells treated with DMSO and FTY720 (15 min).

[0022] FIGS. 11A-11C demonstrate in vivo injection of FTY720 does not decrease C4 RNA. FIGS. 11A-11C show C4 expression in retina (FIG. 11 A), frontal cortex (FIG. 11B), and thalamus (FIG. 11C) of mice treated with DMSO and FTY720. It is theorized that FTY720 inhibits the processing of C4 and its secretion, rather than affecting RNA levels of C4.

[0023] FIGS. 12A-12B indicate the relationship between a complement component and synaptic pruning. FIG. 12A demonstrates that complement component 4 (C4) is associated with a high risk of schizophrenia. FIG. 12B demonstrates that schizophrenic patients have less synapses. See Glantz et al., Arch Gen Psychiatry (2000) 57(l):65-73.

[0024] FIGS. 13A-13B demonstrate the biological function of C4. FIG. 13A shows the complement activation pathways of the complement system, which is an essential component of innate immunity. See Wagner el al. Nature Reviews Drug Discovery (2010) 9:43-56. FIG. 13B demonstrates synapse pruning during development and shows that Clq-/-C3-/-C4-/- mice have less synaptic pruning compared to wile type mice. See Stephan et al. Amur Rev. Neurosci. (2012) 35:369- 389; Sekar et al. Nature (2016) 530(7589): 177-183.

[0025] FIGS. 14A-14D demonstrate the structure expression and association of C4 with schizophrenia. FIG. 14A shows the functional specialization of C4 into C4A and C4B and indicates the sequences differences between C4A and C4B. FIG. 14B shows the structural variation of C4. FIG. 14C provides the measure copy number of each C4 gene type. FIG. 14D shows the schizophrenia risk associated with various structural forms of C4 (left panel) and brain mRNA expression levels associated with various structural forms of C4 (right panel). See Sekar et al. Nature (2016) 530(7589): 177-183. [0026] FIG. 15 demonstrates that reduced synapses number in schizophrenia patients may be explained by excessive synaptic pruning due to increased C4 expression, and that compounds that reduce C4 levels might then rescue the over pruning phenotype.

[0027] FIGS. 16A-16E show where the complement components are produced in the CNS. FIG. 16A shows that astrocytes express and secrete C3. C3 mRNA levels for wild type and IkBa knockout (KO) primary neurons or astroglia are provided, as is ELISA quantification of C3 protein levels in conditioned media of WT or lKBa KO astroglial cultures. See Lian et al. Neuron. (2015) 85(1): 101-115. FIG. 16B shows that astrocytes upregulate Clq expression for all three chains (A, B, and C) by neurons. See Stevens et al, Cell (2007) 131(6): 1164-1178. FIG. 16C shows that astrocytes express C4. FIG. 16D shows genome-wide distributions of expression fidelity for astrocytes (A), oligodendrocytes (O), microglia (M), and neurons (N). See Kelley et al. (2018) oldhamlab.ctec.ucsf.ed. In the CNS neurons, astrocytes, microglia, and oligodendrocytes can synthetize complement components. In fact, astrocytes are able to synthetize as many complement components as the liver. FIG. 16E shows a mixed population of iPSC-derived neurons and astrocytes by immunostaining.

[0028] FIGS. 17A-17B demonstrate that human astrocytes produce and secrete C4. FIG. 17A shows protein expression in 1016A cells by immunostaining.

C4 (green), ALDH1L1 (red) and DAPI (blue) staining of 1016A HA, (scale bar, 20 pm) FIG. 17B shows protein secretion by ELISA. C4 secretion measured by ELISA from 1016A HA astrocytes supernatant treated for 48 hours with DMSO control, Monensin (1 pM) and INFy (250 ng/mL). Data are presented as mean ± SD using Mann Whitney test **** p < 0.0001 (left panel). C4 secretion in primary astrocytes and Hues8 HA treated with vehicle (DMSO) Monensin (1 pM) and INFy (250 ng/mL). Data are presented as mean ± SD using Mann Whitney test **** p < 0.0001 (right panel).

[0029] FIG. 18 demonstrates C4 depletion in astrocytes decreases synaptic C4.

[0030] FIGS. 19A-19F show connecting pathways using the Broad

Connectivity Map (CMap) platform. FIG. 19A provides a schematic of using the CMap platform. FIG. 19B provides a Principal Component analysis (PCA) that highlights three main clusters. FIG. 19C provides correlations between C43 expression and compound signatures. FIG. 19D shows mechanism of action (MO A) of NFKB and P97 inhibitor. FIG. 19E shows that CMap can potentially explain the different potency of BET inhibitors. FIG. 19F demonstrates the proposed intertalk between multiple pathways for the regulation of C4.

[0031] FIGS. 20A-20B demonstrate NFKB inhibitor treatment decreases C4 secretion but not C4A mRNA. FIG. 20A provides a dose response curve of NFKB inhibitor IMD0354 in 1016A derived astrocyte. FIG. 20B provides qPCR of mRNA expression of C4A and C4B upon treatment of 1016A derived astrocyte with

IMD0345 1 uM for 24 and 48 hours.

[0032] FIGS. 21A-21B demonstrate treatment with NFKB inhibitor interferes with pro-inflammatory signals. FIG. 21 A shows inhibition of secretion of C4 from 1016A derived astrocytes upon co-treatment with pro-inflammatory stimuli such as (INFy and Poly I:C) plus or minus IMD0354 1 uM. FIG. 21B shows block of p65 translocation into the nucleus checked by immunofluorescence (upper panel) upon co stimulation with Poly I:C and IMD0354. Lower panel provides quantification of p65 positive nuclei over the total number of nuclei.

[0033] FIGS. 22A-22B demonstrate JAKi decreases C4 RNA as well as interfering with pro-inflammatory stimuli. FIG. 22A provides qPCR of mRNA expression of C4A and C4B upon treatment of 1016A derived astrocyte with JAK inhibitor Tofacitinib 1 uM for 24 and 48 hours. FIG. 22B shows inhibition of secretion of C4 from 1016A derived astrocytes upon co-treatment with pro- inflammatory stimuli such as (INFy and Poly EC) plus or minus the Jak inhibitor (JAKi) Tofacitinib 1 uM.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Astrocytes are crucial for the formation and remodeling of synapses.

As disclosed herein, it is shown that astrocytes are able to secrete the complement component 4 (C4), and that this secretion can be modulated. Disclosed herein are screens for identifying modulators of C4 secretion. The identified modulators of C4 secretion may be involved in the functional rescue of synaptic over-pruning. Also disclosed herein are methods of modulating C4 secretion by administering an agent or compound. Further disclosed herein are methods of treating a neurodegenerative or neuropsychiatric disease by administering an agent that downregulates C4 expression.

[0035] Disclosed herein are methods of screening test agents to identify treatment agents for a disease (e.g., a neurodegenerative, neuropsychiatric or neurodevelopmental disease). In some aspects, astrocytes are treated with a test agent. Examples of test agents that may be screened include those contained within a Target Selective Inhibitor Library (Sellckchem (Catalog No. L3500)), incorporated herein by reference. The astrocytes may be obtained from any source (e.g., the astrocytes may be primary astrocytes or stem cell-derived astrocytes). The level of secreted C4 from the treated astrocytes may be assessed and measured, for example, by using an ELISA or an AlphaLISA. In some aspects, the total number of nuclei in the treated cells is counted and used to normalize the C4 secretion levels. The number of counted nuclei may be compared to a standard deviation of a control (e.g., a DMSO control). In some aspects, the number of nuclei, and thus the level of C4 secretion, is below the standard deviation of the control. In some aspects agents are identified as being of interest as a modulator of C4 secretion if the level of C4 secretion is decreased by up to 10%. A test agent identified as downregulating C4 secretion may exhibit beneficial effects on a disease (e.g., a neurodegenerative, neuropsychiatric or neurodevelopmental disease). In some aspects the test agent may reduce excessive synaptic pruning.

[0036] In some embodiments, the invention provides methods of treating or preventing a disease characterized by over secretion of complement component 4 (C4) comprising administering to the subject an effective amount of at least one agent which modulates C4 secretion in a subject. In some aspects the at least one agent is an agent which decreases or reduces C4 secretion. In some aspects the at least one agent is an agent which modulates (e.g., decreases) the expression of C4. In some aspects the disease characterized by over secretion of C4 is a neurodegenerative, neuropsychiatric, or neurodevelopmental disease. In some aspects the disease characterized by over secretion of C4 exhibits excessive synaptic pruning. In some aspects the disease is schizophrenia. In some aspects the disease is Alzheimer’s disease. In some aspects the disease is Rett Syndrome. In some aspects the disease is Huntington Disease. In some aspects the disease is multiple sclerosis. [0037] In some embodiments the at least one agent which downregulates the over secretion of C4 is an agent which targets one or more pathways involved in C4 secretion. In some aspects, the at least one agent targets an epigenetic pathway, a JAK/STAT pathway, a PI3K/Akt/mTOR pathway, a MAPK pathway, a metabolism pathway, an angiogenesis pathway, a GPCR and/or G protein pathway, a NF-kB pathway, a proteases pathway, a protein tyrosine kinase pathway, an ubiquitin pathway, an apoptosis pathway, a cell cycle pathway, a DNA damage pathway, a transmembrane transporter pathway, an endocrinology and hormone pathway, a neuronal signaling pathway, a TGF-Beta/SMAD pathway, a microbiology pathway, or a cytoskeletal signaling pathway. In some aspects, the at least one agent targets an epigenetic pathway. In some aspects, the at least one agent targets JAK/STAT pathway. In some aspects, the at least one agent targets a PI3K/Akt/mTOR pathway. In some aspects, the at least one agent targets a MAPK pathway. In some aspects, the at least one agent targets a metabolism pathway. In some aspects, the at least one agent targets an angiogenesis pathway. In some aspects, the at least one agent targets a GPCR and/or G protein pathway. In some aspects, the at least one agent targets a NF-kB pathway (e.g., NF-kB inhibitor IMD0354). In some aspects, the at least one agent targets a proteases pathway. In some aspects, the at least one agent targets a protein tyrosine kinase pathway. In some aspects, the at least one agent targets an ubiquitin pathway. In some aspects, the at least one agent targets an apoptosis pathway. In some aspects, the at least one agent targets a cell cycle pathway. In some aspects, the at least one agent targets a DNA damage pathway. In some aspects, the at least one agent targets a transmembrane transporter pathway. In some aspects, the at least one agent targets an endocrinology and hormone pathway. In some aspects, the at least one agent targets a neuronal signaling pathway. In some aspects, the at least one agent targets a TGF-beta/SMAD pathway. In some aspects, the at least one agent targets a microbiology pathway. In some aspects, the at least one agent targets a cytoskeletal signaling pathway. Targeting of a pathway is used to describe an agent that acts as an agonist or antagonist of a pathway.

[0038] In some embodiments the at least one agent which downregulates over secretion of C4 is an agent which targets BET, JAK, Akt, p38 MAPK, histone deacetylase (HD AC), DNA methyltransferase (DNMT), phospholipase A (PLA), ferroptosis, Src, Bruton’s tyrosine kinase (BTK), BMI, PARP, S1P receptor, IkB/IKKI, DPP-4, IGF-1R, p97, Bcl-2, Chk, or PPAR. In some aspects, the at least one agent targets BET. The agent may be a BET bromodomain inhibitor, and in some aspects is an inhibitor of one or more of BRD2, BRD3, and BRD4 (e.g., JQ1 or OTX15). In some aspects, the at least one agent targets JAK. The agent may be a JAK inhibitor (e.g., Tofacitinib), and in some aspects is an inhibitor of one or more of JAK1, JAK2, and JAK3. In some aspects, the at least one agent targets Akt. The agent may be an Akt inhibitor, and in some aspects is an inhibitor of one or more of Aktl, Ak2, and Akt3. In some aspects, the at least one agent targets p38 MAPK. The agent may be a p38 inhibitor, and in some aspects is an inhibitor of one or more of p38a and r38b. In some aspects, the at least one agent targets HD AC. The agent may be an HD AC inhibitor (e.g., an HDAC6 inhibitor). In some aspects, the at least one agent targets DNMT. The agent may be a DNMT inhibitor, and in some aspects is an inhibitor of one or more of DNMT1, DNMT3A, and DNMT3B. In some aspects, the at least one agent targets PLA. The agent may be a PLA inhibitor (e.g., a non-pancreatic secretory PLA2 inhibitor). In some aspects, the at least one agent targets ferroptosis. The agent may be a ferroptosis activator. In some aspects, the at least one agent targets Src. The agent may be a Src inhibitor (e.g., a dual Src/Abl inhibitor). In some aspects, the at least one agent targets BTK. The agent may be a Btk inhibitor. In some aspects, the at least one agent targets BMI. The agent may be a BMI inhibitor (e.g., a BMI-l inhibitor). In some aspects, the at least one agent targets PARP. The agent may be a PARP inhibitor (e.g., a PARP1 inhibitor). In some aspects, the at least one agent targets S1P receptor. The agent may be a S1P antagonist (e.g., FTY720). In some aspects, the at least one agent targets IkB/IKKI. The agent may be an inhibitor of IKK (e.g., an IKKb inhibitor). In some aspects, the at least one agent targets DPP-4. The agent may be a DPP-4 inhibitor. In some aspects, the at least one agent targets IGF-1R. The agent may be a IGF-1R inhibitor. In some aspects, the at least one agent targets p97. The agent may be a p97 inhibitor (e.g., NMS-873). In some aspects, the at least one agent targets Bcl-2. The agent may be a Bcl-2 inhibitor. In some aspects, the at least one agent targets Chk. The agent may be a Chk inhibitor, and in some aspects is an inhibitor of one or more of Chkl and Chk2. In some aspects, the at least one agent targets PPAR. The agent may be an antagonist of PPAR (e.g., a PPARy antagonist). [0039] As used herein, the term“treating” and“treatment” refers to administering to a subject an effective amount of a composition so that the subject as a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term“treatment” includes prophylaxis. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted.

“Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0040] By“treatment, prevention or amelioration of neurodegenerative disorder” is meant delaying or preventing the onset of such a disorder, at reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the progression or severity of such a condition. In one embodiment, the symptom of a disorder characterized by over secretion of C4 is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of a disorder characterized by over secretion of C4 is alleviated by more than 50%. In one embodiment, the symptom of a disorder characterized by over secretion of C4 is alleviated by 80%, 90%, or greater. In one embodiment, the symptom of a neurodegenerative disorder is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of a neurodegenerative disease is alleviated by more than 50%. In one embodiment, the symptom of a neurodegenerative disorder is alleviated by 80%, 90%, or greater. In some embodiments, treatment also includes improvements in synaptic function. In some embodiments, synaptic function improves by at least about 10%, 20%, 30%, 40%, 50% or more. In some embodiments, treatment includes downregulating the secretion of C4. In some embodiments, the secretion of C4 is reduced by at least about 10%, 20%, 30%, 40%, 50% or more. [0041] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and“subject” are used interchangeably herein.

[0042] In some embodiments, the subject is a human.

[0043] The term“test compound” refers to any of a small molecule, nucleic acid, amino acid, polypeptide, antibody and antibody-like molecules, aptamers, macrocycles, or other molecules. In certain embodiments, a test compound is a small organic molecule. In one aspect of these embodiments, the small organic molecule has a molecular weight of less than about 5,000 daltons.

[0044] As used herein,“neuro disorder” or“neuro disease” refer to neurodegenerative disorders, neuropsychiatric disorders and/or neurodevelopmental disorders. Neuro disorders may be any disease affecting neuronal network connectivity, synaptic function and activity. “Neurodegenerative disorder” refers to a disease or disorder caused by or associated with the deterioration of cells or tissues of the nervous system. Non-limiting examples of neurodegenerative disorders include poly glutamine expansion disorders (e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and

spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado- Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e.g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer-Vogt- Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Guillain-Barre syndrome, ischemia stroke, Krabbe disease, kuru, Lewy body dementia, multiple sclerosis, multiple system atrophy, non-Huntingtonian type of Chorea, Parkinson's disease, Pelizaeus- Merzbacher disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), SteeleRichardson-Olszewski disease, and Tabes dorsalis.

[0045] In certain contexts, neurodegenerative disorders encompass neurological injuries or damages to the CNS or the PNS associated with physical injury (e.g., head trauma, mild to severe traumatic brain injury (TBI), spinal cord injury, diffuse axonal injury, craniocerebral trauma, cranial nerve injuries, cerebral contusion, intracerebral haemorrhage and acute brain swelling), ischemia (e.g., resulting from spinal cord infarction or ischemia, ischemic infarction, stroke, cardiac insufficiency or arrest, atherosclerotic thrombosis, ruptured aneurysm, embolism or haemorrhage), certain medical procedures or exposure to biological or chemic toxins or poisons (e.g., surgery, coronary artery bypass graft (CABG), electroconvulsive therapy, radiation therapy, chemotherapy, anti-neoplastic drugs, immunosuppressive agents, psychoactive, sedative or hypnotic drugs, alcohol, bacterial or industrial toxins, plant poisons, and venomous bites and stings), tumors (e.g., CNS metastasis, intraaxial tumors, primary CNS lymphomas, germ cell tumors, infiltrating and localized gliomas, fibrillary astrocytomas, oligodendrogliomas, ependymomas, pleomorphic xanthoastrocytomas, pilocytic astrocytomas, extraaxial brain tumors, meningiomas, schwannomas, neurofibromas, pituitary tumors, and mesenchymal tumors of the skull, spine and dura matter), infections (e.g., bacterial, viral, fungal, parasitic or other origin is selected from the group consisting of pyrogenic infections, meningitis, tuberculosis, syphilis, encephalomyelitis and leptomeningitis), metabolic or nutritional disorders (e.g., glycogen storage diseases, acid lipase diseases, Wernicke's or Marchiafava-Bignami's disease, Lesch-Nyhan syndrome, Farber's disease, gangliosidoses, vitamin B12 and folic acid deficiency), cognition or mood disorders (e.g., learning or memory disorder, bipolar disorders and depression), and various medical conditions associated with neural damage or destruction (e.g., asphyxia, prematurity in infants, perinatal distress, gaseous intoxication for instance from carbon monoxide or ammonia, coma, hypoglycaemia, dementia, epilepsy and hypertensive crises). [0046] In some embodiments, the subject suffers from a disorder or disease characterized by over secretion of C4. In some aspects the subject suffers from a disorder or disease characterized by excessive synaptic pruning. In some aspects the subject suffers from a neurodegenerative disease. In some aspects the

neurodegenerative disease is characterized by over secretion of C4 or excessive synaptic pruning.

[0047] In some embodiments the methods described herein further comprise selecting a subject diagnosed with a disorder characterized by over secretion of C4. In some aspects the methods described herein further comprise selecting a subject diagnosed with a disorder characterized by excessive synaptic pruning. In some aspects the methods described herein further comprise selecting a subject diagnosed with a neurodegenerative disease. A subject suffering from a neurodegenerative disease can be selected based on the symptoms presented.

[0048] In some embodiments, the methods described herein further comprise diagnosing a subject for a neurodegenerative disease or disorder. In some embodiments, the methods described herein further comprise diagnosing a subject for schizophrenia. In some embodiments, the methods described herein further comprise diagnosing a subject for Alzheimer’s disease.

[0049] In some embodiments the methods further comprises co-administering an additional pharmaceutically active agent approved for treatment of the neurodegenerative disorder or alleviating a symptom thereof.

[0050] EXAMPLE 1:

[0051] Screening and hit selection

[0052] 96 wells m-clear black imaging plates (Grainer #655090) were coated with Poly-L-Lysine (MP #0215017610) at a final concentration of 15 pg/mL using Biotek. Plates were incubated overnight at 37 °C. The day after plates were washed 3 times with PBS and AM media was added to each well. Coating washes and media addition was performed using the Biotek. iPSC-derived astrocytes were plated at a concentration of 3X10⁵ cells per well using the Multidrop™ Combi Reagent Dispenser. The day after the media was replaced with fresh media and compounds were added at two different concentrations 1 and 0.3 mM using the Thermo Scientific Matrix Hydra II 96-Channel Automated Liquid Handling System. The screening was performed in triplicate plates. The small molecule Target Selective Inhibitor library was purchased from Sellckchem (L3500). Two days after the addition of the compounds the supernatant was used to perform ELISA (additional details below) and plates were stained with Hoechst using the Multidrop™ Combi Reagent Dispenser and quantified using Operetta High-content imaging system from PerkinElmer. Nuclei were counted using Columbus Image Data Storage and Analysis System (Perkin Elmer). The total number of nuclei was used to normalize the C4 secretion levels. Number of nuclei that were 3 times below the standard deviation of DMSO control were excluded from further analysis to exclude toxic and thus potential false positive. Compounds identified as hits for further consideration were those compounds that decrease the secretion of C4 up to 10%. Compounds identified as being of interest (i.e., hits) are included in Table 1. Twenty-five compounds with greater C4 downregulation or interesting pathways were selected for a secondary validation (FIG. 2C). The secondary screening was performed as previously described. Cherry picked compounds from the stock library or freshly purchased compounds were tested at in 4 concentrations (3, 1, 0.3 and 0.1 mM in triplicates). 23 out of the 25 compounds tested were validated.

[0053] Table 1 : Compounds of interest

[0054] Materials and Methods

[0055] Human pluripotent stem cell culture

[0056] iPSCs and ESCs were cultured in StemFlex medium (ThermoFisher A3349401). When pluripotent stem cells reached 80-85% of confluency, colonies were dissociated using 0.5 mM EDTA in calcium/magnesium-free PBS at room temperature and passaged on Matrigel (Coming 354234) coated 10 or l5-cm2 tissue culture dishes (Coming). All human pluripotent stem cells used were maintained below passage 40 and confirmed to be karyotypically normal and mycoplasma negative.

[0057] Bright field images and immunofluorescence

[0058] Astrocytes were plated on PLL coated plates 6 wells or 96 wells at a density of 5X10⁵ cells and 3X10⁴ cells per well respectively. The next day cells were fixed using 4% PFA for 15 minutes and washed with PBS three times. The cells were blocked in 10% horse serum 0.01% Triton X-100 in PBS (for CD44 staining only) or 5% horse serum 0.3% PBS Triton X-100 for 1 hour a room temperature. Primary antibodies were diluted in 5% horse serum at 4°C overnight followed by washes in PBS and incubation with secondary antibodies (diluted 1: 1000) and Hoechst (1:5000) for 1 hour a room temperature. Fluorescently conjugated antibodies used were goat anti-mouse IgG Alexa Fluor 488 (Life Technologies Al 1001) goat anti-rabbit IgG Alexa Fluor 546 (Life Technologies A11010). Bright field images were acquired using an inverted Eclipse Ti microscope (Nikon). Immunofluorescences were acquired either using a ImageXpress Micro Confocal (Molecular device) or Opera High Content Screening System (Perkin Elmer). All images were processed with Adobe Photoshop software.

[0059] Flow Cvtometrv Analysis

[0060] Freshly dissociated or frozen astrocytes were cultured as previously described until they reached 80% confluency. Cells were detached using Trypsin- EDTA solution (Sigma, T3924). 1X10⁶ cells were stained following the

manufacturer's instruction for cell surface antigens using directly conjugated antibodies against FITC CD44, (555478), CD200 PerCP-Cy5.5 (562124) or isotypes control FITC Mouse igG2B k (555742) and Per CP-Cy 5.5 IgGl k (550795). All antibodies were purchased form BD Pharmingen. Hoechst (1:5000) was used as viability markers. Samples were analyzed on the LSRII flow cytometer (BD

Biosciences, San Diego), and data was analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

[0061] ELISA

[0062] All washes were performed using 150 ul of PBS containing 0.05% Tween for three times. All incubations were performed at 37°C unless otherwise specified. Antibodies were incubated in a volume of 50 ul per well. 96 well plates (Thermo Scientific 439454) were coated (overnight at 4°C) with goat anti human C4 antibody (Quidel A305 1: 1000) in PBS. The day after the plates were washed and incubated with blocking solution (1% BSA in PBS) for 1 hour. After elimination of the blocking solution 80 uL of astrocytes supernatant was added to each well and incubated for 1 hour and 30 minutes. Following washed the samples were incubated with a rabbit anti human C4 Ab (Dako F 0169 1 : 3000) for one hour. Following washed the plates were incubated for 30 minutes with goat-anti-rabbit Alkaline Phosphatase (abeam ab97048). In the last step following washes the plates were incubated with 1 M diethanolamine buffer, 0.5 mM MgCl2, pH 9.8 containing Phosphatase substrate (Sigma S0942). The reaction was stopped with 3 N NaOH and read at 405 nm using Molecular Devices SpectraMax M5 Reader. As a reference for quantification, a standard curve was established by a serial dilution of purified human complement protein C4 (Quidel A402) starting from 100 ng/mL.

[0063] Cytokines array [0064] Astrocytes were plated in 6 well plates at a density of 5X10⁵ cells per well in complete AM media (Science Cell) the next day cells were treated with compound or mock and after 48 hours supernatant was collected and stored at-80 °C.

[0065] Proteome Profiler™ Human Cytokine Array (R&D Systems,

#ARY005B) was used according to manufacturer's guidelines. Proteome profiler intensity dot blots were quantified using Adobe Photoshop software and were normalized to mean intensities of reference spots.

[0066] Cell Lysis and western Blot

[0067] Cells were lysate in RIPA buffer (Sigma Aldrich R0278) with protease inhibitors (Termo Fisher 78426) and phosphatase inhibitors (78426). Whole cell lysate were loaded on NuPAGE 4-12% Bis-Tris gels (Life technologies) and transferred to poly vinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). After incubation with 5% milk in TBST (TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 hour. The membrane was washed once with TBST and incubated with antibodies against C4 (Dako F 0169 1 : 1000 or Quidel A305 1 : 1000 in 5% TBST overnight at 4°C. Membranes were washed three times for 10 minutes and incubated with a 1: 1000 dilution of horseradish peroxidase-conjugated anti-rabbit antibodies for 1 h. Blots were washed with TBST three times and developed with the SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific 34075).

[0068] Chromatin purification and WB

[0069] Cells were cultured and treated with DMSO control or (+)- JQ1 for 24 hours. Cells were collected using trypsin-EDTA solution, washed once with cold PBS and fast-frozen. The Chromatin bound fraction and the cytoplasmic fraction were isolated using ThermoFisher’s Kit (78840) following manufacturer’s instruction. Equal amount of proteins (5 pg) were loaded on a gel as previously described.

Antibody incubation was done overnight in 5% BSA in T-BST at 4 °C with gentle agitation, BRD4 1 :200 (Abeam 128874), Histone H3 1:20000 (Cell Signaling 9715) and b-Actin (Cell Signaling 8H10D10).

[0070] qRT- PCR

[0071] RNA was extracted using the RNeasy mini Kit (QIAGEN). cDNA was prepared with iScript cDNA Synthesis Kit Bio (Bio-Rad 170-8891). All quantitative RT-PCR reactions were performed in triplicate using Fast SYBR Green Master Mix (Thermos Fisher Scientific 4385614) and data was acquired on the AB7900HT detection system (Applied Biosystems). Ct values were calculated and normalized to GAPDH and the relative expression ratio was calculated using the Pfaffl method (Pfaffl, 2001 ). KiCqStart™ Primers were purchased from Sigma (available upon request).

[0072] EXAMPLE 2:

[0073] Astrocytes play a critical role in CNS function and disease. Due to the fundamental differences between mouse and human astrocytes, accurate disease modeling requires the use of human cells. To this end, a new bioreactor protocol has been developed for producing large numbers of astrocytes from pluripotent cells. The astrocytes express canonical cell markers of astrocytes and are functional. Next a pilot ELISA-based screen was performed, looking for small molecule modulators of astrocyte secretion of complement component 4, a gene recently associated with an increased risk of schizophrenia. A previously described astrocyte production method was modified to make it more compatible with a variety of in vitro and in vivo uses. Further, an ELISA assay is transitioned to an AlphaLISA format to allow for large- scale high-throughput screening.

[0074] Background and significance

[0075] Astrocytes play a critical role in synapse formation, function and elimination (Chaboub and Deneen 2013). Recently, their contribution to

neuropsychiatric and neurodegenerative diseases has been increasingly

acknowledged. Importantly, several studies have highlighted the differences in morphology, physiology and complexity between mouse and human astrocytes (Windrem, Osipovitch et al. 2017). Thus, studying how astrocytes are involved in human central nervous system (CNS) diseases will require the use of patient-specific astrocytes.

[0076] Current protocols for the directed differentiation of astrocytes from human pluripotent stem cells typically require an extensive culture period (up to 6 months) (Krencik, Weick et al. 2011, Palm, Bolognin et al. 2015, Dezonne, Sartore et al. 2017, Sloan, Darmanis et al. 2017) and rely on the ability to generate and isolate intermediate progenitors (Krencik, Weick et al. 2011, Shaltouki, Peng et al. 2013, Santos, Vadodaria et al. 2017, Tew, Wang et al. 2017, Lundin, Delsing et al. 2018). To circumvent these problems, a new bioreactor-based protocol for rapid, reproducible and large-scale production of human astrocytes has been established. These cells express canonical astrocyte markers and they are able to secrete different cytokines as well as complement components, such as complement component 4 (C4) (Levi-Strauss and Mallat 1987, Bamum 1995, Choi, Lee et al. 2014, Tew, Wang et al. 2017). Importantly, this protocol is unique in that a population of astrocytes is produced where the vast majority of cells are GFAP -negative, consistent with the generation of normal, non-reactive astrocytes (Liddelow, Guttenplan et al. 2017).

[0077] This pure population of astrocytes was used to conduct an ELISA- based small molecule screen to identify and validate compounds able to decrease complement C4, a key member of the complement cascade. In the CNS, the complement pathway plays a role in synaptic pruning, a physiological process that occurs during normal brain development, extending to the teenage years (Stevens, Allen et al. 2007). Excessive pruning has been shown to result in neurodegenerative and neuropsychiatric disease. In particular, recent research has shown that variation in the copy number and expression of C4 strongly correlates with the development of schizophrenia (Schizophrenia Working Group of the Psychiatric Genomics 2014, Sekar, Bialas et al. 2016).

[0078] Since the discovery of dopamine receptor blockers decades ago, no effective drugs for schizophrenia have been identified (Carlsson and Lindqvist 1963, Lieberman 2007). A new possibility, suggested by recent research, is targeted inhibition of the complement system. The discovery of compounds that can regulate complement will be of broad therapeutic interest in multiple diseases in which an impairment of cognition due to loss of synapses is observed. To that end, a small molecule screen was performed that lead to identification of several compounds able to decrease the secretion of C4. Among the identified hits, the mechanism of action of an epigenetic modifier, the bromodomain inhibitor (+)-JQl, was explored. Treatment of astrocytes with (+)-JQl decreased several critical complement components (Clq, C3 and C4) via depletion of BRD4 on chromatin. Moreover, (+)-JQl was also able to suppress pro-inflammatory cytokine secretion maintaining the astrocytes in a non reactive state. Next it was shown that this treatment culminates in the decline of synaptic C4 in stem cell-derived cortical neurons. Lastly, in vivo treatment of mice with (+)-JQl significantly reduced C4 mRNA in the pre-frontal cortex, a key area for higher cognitive function.

[0079] It is desired to improve the yield and efficiency of the current differentiation protocol using defined culture conditions. In addition, it would be beneficial to establish a high-throughput AlphaLISA that will enable large-scale screening to allow the identification of BBB-penetrant compounds that reduce brain C4 levels. These improvements will allow the described platform to be used broadly for drug discovery as well as for cell transplant therapy.

[0080] Improve the yield and efficiency of the current differentiation protocol using defined culture conditions.

[0081] Although the current protocol is very efficient in generating a pure population of astrocytes, it requires the transition from 3D to 2D culture that leads to a dramatic decrease in cell numbers as the cells are passaged and switched to an expensive commercially-sourced medium of unknown composition. This makes the protocol suboptimal for large-scale cell production and clinical use.

[0082] The current protocol requires 30 days of differentiation in 3D spinner flasks to generate a pure population of astrocytes. After 30 days, cells are dissociated and cryopreserved or cultured in standard tissue culture flasks in an undefined astrocyte medium (AM) from ScienCell. This medium contains Fetal Bovine Serum (FBS) and other undisclosed growth supplements (AGS). To avoid the use of the commercial medium, especially one that contains xeno-materials, different basal media (such as DMEMF-12 or Neurobasal) and serum replacements will be tested. To support the growth and expansion of astrocytes, while maintaining their non-reactive nature, a combination of cytokines (CTNF, HB-EGF and others) will be tested. Once a defined medium is identified this will be used during the final 10 days of 3D astrocyte culture, allowing the cells to adapt. This pre-exposure to the defined medium in 3D should increase the recovery of cells after dissociation and plating in 2D. In addition, plating on 3D spheres on poly-L-Lysine coated plates will be tested to allow these cells to naturally migrate outside of the spheres, avoiding the stressful mechanical dissociation process. The evaluation of the improvements in the plating methods will be done by comparing the survival and the rate of proliferation of cells compared to previous methods. The purity and the quality of the cells will be assessed by FACS and staining for canonical markers of astrocytes, compared to what is found with current methods.

[0083] Establish a high-throughput AlphaLISA that will enable large-scale screening to allow the identification of BBB-penetrant compounds that reduce brain C4 levels.

[0084] The first set of data obtained from a screen of about 500 compounds clearly shows the robustness of the described assay in identifying compounds able to decrease secreted C4. However, the current ELISA format cannot readily

accommodate larger-scale screens.

[0085] It is desired to optimize the assay for high-throughput screening (HTS). This can be achieved by converting the conventional ELISA into an Amplified Luminescent Proximity Homogenous Assay (AlphaLISA) (PerkinElmer). As shown in other HTS campaigns, this technology allows for fast, straightforward (no washes needed) and more sensitive target detection (Chan, Cottrell et al. 2014). The initial step will be to test the sensitivity of the AlphaLISA to ensure that it can measure secreted C4. The second step will involve the optimization and miniaturization of the assay to get a fast, cost-effective, and robust screening platform. To validate this platform, a library of about 300 BBB penetrant compounds will be screened

(MedChemExpress) using astrocytes derived from a schizophrenic patient with a high copy number of C4. Hits that show significant C4 downregulation will be selected and validated, and 5-10 compounds will be selected for careful dose-response curves. If any promising candidates are found, in vitro and in vivo functional synaptic pruning experiments will be performed, as is currently being done with existing hit compounds.

[0086] References

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Claims

CLAIMS What is claimed is:

1. A method of treating a neurodegenerative and/or neuropsychiatric disorder comprising administering to a subject an agent that modulates secretion of complement component 4 (C4), thereby treating a neurodegenerative and/or neuropsychiatric disorder in the subject.

2. The method of claim 1, wherein the agent downregulates secretion of C4.

3. The method of claim 1 or 2, wherein the agent targets one or more pathways involved in C4 secretion.

4. The method of any one of claims 1-3, wherein the neurodegenerative and/or neuropsychiatric disorder is selected from the group consisting of Alzheimer’s Disease, Rett Syndrome, Huntington Disease, Multiple Sclerosis, and

Schizophrenia.

5. The method of any one of claims 1-4, wherein the agent targets BET.

6. The method of claim 5, wherein the agent is a BET bromodomain inhibitor.

7. The method of claim 5 or 6, wherein the agent is an inhibitor of one or more of

BRD2, BRD3, and BRD4.

8. The method of any one of claims 1-4, wherein the agent targets JAK.

9. The method of claim 8, wherein the agent is a JAK inhibitor.

10. The method of claim 8 or 9, wherein the agent is an inhibitor of one or more of

JAK1, JAK2, and JAK3.

11. The method of any one of claims 1 -4, wherein the agent targets Akt.

12. The method of claim 11, wherein the agent is an Akt inhibitor.

13. The method of claim 11 or 12, wherein the agent is an inhibitor of one or more of Aktl, Akt2, and Akt3.

14. The method of any one of claims 1-4, wherein the agent targets p38 MAPK.

15. The method of claim 14, wherein the agent is a p38 inhibitor.

16. The method of claim 14 or 15, wherein the agent is an inhibitor of one or more of p38a and r38b.

17. The method of any one of claims 1-4, wherein the agent targets histone

deacetylase (HD AC).

18. The method of claim 17, wherein the agent is an HD AC inhibitor.

19. The method of claim 17 or 18, wherein the agent is an HDAC6 inhibitor.

20. The method of any one of claims 1-4, wherein the agent targets DNA

methyltransferase (DNMT).

21. The method of claim 20, wherein the agent is a DNMT inhibitor.

22. The method of claim 20 or 21, wherein the agent is an inhibitor of one or more of DNMT1, DNMT3A, and DNMT3B.

23. The method of any one of claims 1-4, wherein the agent targets phospholipase A (PLA).

24. The method of claim 23, wherein the agent is a PLA inhibitor.

25. The method of claim 23 or 24, wherein the agent is a non-pancreatic secretory phospholipase A2 inhibitor.

26. The method of any one of claims 1-4, wherein the agent targets ferroptosis.

27. The method of claim 26, wherein the agent is a ferroptosis activator.

28. The method of any one of claims 1-4, wherein the agent targets Src.

29. The method of claim 28, wherein the agent is a Src inhibitor.

30. The method of claim 28 or 29, wherein the agent is a dual Src/Abl inhibitor.

31. The method of any one of claims 1-4, wherein the agent targets Bruton’s tyrosine kinase (BTK).

32. The method of claim 31, wherein the agent is a Btk inhibitor.

33. The method of any one of claims 1-4, wherein the agent targets BMI.

34. The method of claim 33, wherein the agent is a BMI inhibitor.

35. The method of claim 33 or 34, wherein the agent is a BMI-l inhibitor.

36. The method of any one of claims 1-4, wherein the agent targets PARP.

37. The method of claim 36, wherein the agent is a PARP inhibitor

38. The method of claim 36 or 37, wherein the agent is a PARP1 inhibitor.

39. The method of any one of claims 1-4, wherein the agent targets S1P receptor.

40. The method of claim 39, wherein the agent is a S1P antagonist.

41. The method of any one of claims 1-4, wherein the agent targets IkB/IKKI.

42. The method of claim 41, wherein the agent is an inhibitor of IKK.

43. The method of claim 41 or 42, wherein the agent is an IKKb inhibitor.

44. The method of any one of claims 1-4, wherein the agent targets DPP-4.

45. The method of claim 44, wherein the agent is a DPP-4 inhibitor.

46. The method of any one of claims 1-4, wherein the agent targets IGF-1R.

47. The method of claim 46, wherein the agent is an IGF-1R inhibitor.

48. The method of any one of claims 1-4, wherein the agent targets p97.

49. The method of claim 48, wherein the agent is a p97 inhibitor.

50. The method of any one of claims 1-4, wherein the agent targets Bel -2.

51. The method of claim 50, wherein the agent is a Bcl-2 inhibitor.

52. The method of any one of claims 1-4, wherein the agent targets Chk.

53. The method of claim 52, wherein the agent is a Chk inhibitor.

54. The method of claim 52 or 53, wherein the agent is an inhibitor of one or more of Chkl and Chk2.

55. The method of any one of claims 1-4, wherein the agent targets PPAR.

56. The method of claim 55, wherein the agent is an antagonist of PPAR.

57. The method of claim 55 or 56, wherein the agent is a PPARy antagonist.

58. The method of any one of claims 1-4, wherein the agent targets an epigenetic pathway.

59. The method of any one of claims 1-4, wherein the agent targets a JAK/STAT pathway.

60. The method of any one of claims 1-4, wherein the agent targets a

PI3K/Akt/mTor pathway.

61. The method of any one of claims 1-4, wherein the agent targets a MAPK

pathway.

62. The method of any one of claims 1-4, wherein the agent targets a metabolism pathway.

63. The method of any one of claims 1-4, wherein the agent targets an

angiogenesis pathway.

64. The method of any one of claims 1-4, wherein the agent targets a GPCR

and/or G protein pathway.

65. The method of any one of claims 1-4, wherein the agent targets a NF-kB pathway.

66 The method of any one of claims 1-4, wherein the agent targets a proteases pathway.

67. The method of any one of claims 1-4, wherein the agent targets a protein tyrosine kinase pathway.

68 The method of any one of claims 1-4, wherein the agent targets an ubiquitin pathway.

69. The method of any one of claims 1-4, wherein the agent targets an apoptosis pathway.

70. The method of any one of claims 1-4, wherein the agent targets a cell cycle pathway.

71. The method of any one of claims 1-4, wherein the agent targets a DNA

damage pathway.

72. The method of any one of claims 1-4, wherein the agent targets a

transmembrane transporter pathway.

73. The method of any one of claims 1-4, wherein the agent targets an

endocrinology and hormone pathway.

74. The method of any one of claims 1-4, wherein the agent targets a neuronal signaling pathway.

75. The method of any one of claims 1-4, wherein the agent targets a TGF- beta/SMAD pathway.

76. The method of any one of claims 1-4, wherein the agent targets a

microbiology pathway.

77 The method of any one of claims 1-4, wherein the agent targets a cytoskeletal signaling pathway.

78. The method of any one of claims 1-4, wherein the agent is IMD0354.

79. The method of any one of claims 1-4, wherein the agent is Tofacitinib.

80. The method of any one of claims 1-4, wherein the agent is selected from Table 1

81. The method of any one of claims 1-80, wherein the neurodegenerative and/or neuropsychiatric disorder is schizophrenia.

82. The method of any one of claims 1-80, wherein the neurodegenerative and/or neuropsychiatric disorder is Alzheimer’s Disease.

83. The method of any one of claims 1-3, wherein the modulation of C4 secretion results in the rescue of synaptic over-pruning.

84. A method of modulating secretion of complement component 4 (C4)

comprising administering an agent, wherein the agent is a downregulator of C4 secretion.

85. A method of modulating secretion of complement component 4 (C4)

comprising administering an agent, wherein the agent targets one or more pathways involved in C4 secretion.