Inhibition of CD9 expressing microglia to prevent post-traumatic brain injury cognitive impairment

Abstract

CD9 expressing microglia are observed in various human neurodegenerative diseases beyond traumatic brain injuries, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. CD9 blocking and FcγRIII blocking methods can be widely used as an intervention strategy to prevent disease-associated cognitive impairment. Therefore, disclosed herein are methods for treating a traumatic brain injury in a subject in need thereof that involve the step of administering to the subject a therapeutically effective amount of a composition comprising an anti-CD9 or an anti FcγRIII blocking agent, such as a blocking antibody.

Description

TH Docket No. 222120-2130INHIBITION OF CD9 EXPRESSING MICROGLIA TO PREVENT POST- TRAUMATIC BRAIN INJURY COGNITIVE IMPAIRMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 685,845, filed August 22, 2024, which is incorporated herein by reference in its entirety.CROSS REFERENCE TO SEQUENCE LISTING

[0002] The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The sequence listing in xml format is provided as a file named “222120- 2130_Sequence_Listing.xml” created on August 11 , 2025, and having a size of 6,336 bytes, is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Microglia are resident immune cells in the central nervous system (Crotti, et al. Immunity 201644:505-515; Hammond, et al. Annu Rev Cell Dev Biol 2018 34:523-544; Prinz, et al. Annu Rev Immunol 2021 39:251-277). Microglia react to infection and tissue destruction by recognizing danger molecules, secrete various inflammatory mediators, perform phagocytosis, and facilitate the infiltration of other immune cells. These reactive microglia also lose their homeostatic function to support neurons. Consequently, reactive microglia dysregulate neuronal activities and damage neurons. Recent studies have revealed diverse molecular phenotypes of reactive microglia under various pathological conditions, such as disease-associated microglia (DAM) - first described in mouse models carrying Alzheimer’s disease-related mutations (Keren-Shaul, et al. Cell 2017 169:1276-1290. e1217; Butovsky, et al. Nat Rev Neurosci 2018 19:622-635; Masuda, et al. Cell Rep 2020 30:1271-1281). Nevertheless, the impact of specific reactive microglial phenotypes on neurons and cognitive functions seems highly context dependent. Reactive microglia may be neurodegenerative or neuroprotective.

[0004] Traumatic brain injury (TBI) is a risk factor for the later development of cognitive impairment and dementia (Perry, et al. J Neurosurg 2016 124:511-526; Crane, et al. JAMA Neurol 2016 73:1062-1069; Nordstrom, et al. PLoS Med 2018 15:e1002496; Barnes, et al. JAMA Neurol 2018 75:1055-1061 ; Fann, et al. Lancet Psychiatry 2018 5:424-431 ; Griesbach, et al. J Neurotrauma 2018 35:17-24). In the acute phase after TBI, reactive microglia contribute toTH Docket No. 222120-2130 the maintenance of tissue integrity in the primary lesion: they change morphologies, produce neurotrophic factors, and anti-inflammatory cytokines, and remove cellular debris via phagocytosis (Simon, et al. Nat Rev Neurol 2017 13:171-191 ; Jassam, et al. Neuron 2017 95:1246-1265; Burda, et al. Neuron 2014 81 :229-248). In a more chronic phase, reactive microglial changes are observed in multiple brain regions, including the hippocampus, thalamus, corpus callosum, putamen, supramarginal gyrus, and amygdala (Simon, et al. Nat Rev Neurol 2017 13:171-191 ; Ramlackhansingh, et al. Ann Neurol 2011 70:374-383; Loane, et al. J Neuropathol Exp Neurol 2014 73:14-29). Notably, both human and rodent studies showed highly reactive changes of microglia in the thalamus in a chronic phase of TBI (Ramlackhansingh, et al. Ann Neurol 2011 70:374-383; Loane, et al. J Neuropathol Exp Neurol 2014 73:14-29). Human neuroimaging studies further indicated that thalamic microglial changes correlated with cognitive impairment (Ramlackhansingh, et al. Ann Neurol 2011 70:374-383). Nevertheless, the molecular mechanisms underlying microglial changes in a chronic phase of TBI and their impact on neuropathology and cognitive impairment remain elusive.SUMMARY OF THE INVENTION

[0005] CD9 expressing microglia are observed in various human neurodegenerative diseases beyond traumatic brain injuries, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. CD9 blocking method can be widely used as an intervention strategy to prevent disease-associated cognitive impairment.

[0006] Therefore, disclosed herein are methods for treating a traumatic brain injury in a subject in need thereof that involve the step of administering to the subject a therapeutically effective amount of a composition comprising an anti-CD9 blocking agent, such as a blocking antibody.

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0008] FIGs. 1A to 1 N show cortical brain injuries induce reactive changes in the thalamic microglia and impair recognition memory deficits via thalamic neuronal dysfunction. FIG. 1A contains representative images of Iba1+ microglia in the cortex (primary lesion) and the thalamus 3 and 21 days after unilateral cortical ablation injury and sham surgery. MD, mediodorsal nucleus; CM, central medial nucleus; VPL, ventral posterolateral nucleus; VPM, Ventral posteromedial nucleus. Scale bar, 500 pm. FIG. 1 B shows quantification of area covered by I ba1 + microglia (%) in the cortex and thalamus (n = 3-4 mice for sham; n = 3-7 miceTH Docket No. 222120-2130 for injury). FIG. 10 contains representative images of TNF-a and CD68 expression in Iba1 + microglia in the thalamus 21 days after cortical brain injuries and sham surgery. Scale bar, 10 pm. FIG. 1 D shows quantification of the number of TNF-a expressing Iba1+ microglia and average CD68+ area in Iba1+ microglia (n = 3 mice for sham; n = 3 mice for injury). FIG. 1 E is an experimental timeline for novel object recognition (NOR) test after cortical brain injuries. FIG. 1 F shows discrimination index in the NOR test for injury (n = 6) and sham (n = 7 mice) group. FIG. 1G is a correlation plot of the discrimination index and thalamic Iba1 intensity 21 days after injuries and sham surgery (n = 7 mice). FIG. 1 H contains representative images of NeuN+ neurons in the intact thalamus and 7 and 21 days after injuries. Scale bar, 10 pm. FIG. 11 shows quantification of the number of NeuN+ neurons in the thalamus (n = 3 mice per time point). FIG. 1 J contains representative images of neuronal c-Fos expression in the thalamus, PRh, mPFC, and hippocampus (CA1 , CA3) 21 days after injuries and sham surgery. Scale bars, 100 pm. FIG. 1 K shows quantification of neuronal c-Fos expression in the injury group (n = 6-7 mice) relative to the sham group (n = 3-4 mice). (L) Experimental timeline for neuronal DREADD rescue experiment. AAV-hSyn-hM3D(Gq)-mCherry was injected into the thalamus on the day of cortical brain injuries, and CNO or vehicle was administered intraperitoneally before the test session in the NOR test. FIG. 1M shows quantification of neuronal c-Fos expression in the CNO-treated group (n = 8 mice) relative to the vehicle-treated mice (n = 11 mice). FIG. 1 N shows discrimination index in the NOR test for vehicle-treated (n = 11 mice) and CNO-treated mice (n = 8 mice). Data are shown as the mean ± s.e.m. *p < 0.05, **p < 0.01 , ***p < 0.001 ; Student’s t-test (FIGs. 1 B, 1 D, 1 F, 1 K, 1M, and 1 N) and one-way ANOVA with post hoc Dunnett’s test (FIG. 11).

[0009] FIGs. 2A to 2K shows reactive changes in thalamic microglia are necessary and sufficient to impair neuronal function and recognition memory. FIG. 2A is an experimental timeline for local depletion of thalamic microglia by anti-CSF1 R Ab injection. FIG. 2B shows discrimination index in the NOR test for control Ab group (n = 7 mice) and anti-CSF1 R Ab group (n = 10 mice) after cortical brain injuries. FIG. 2C contains representative images of neuronal c- Fos expression in the thalamus, PRh, mPFC, and hippocampus in control Ab- and anti-CSF1 R Ab-injected group after cortical brain injuries. Scale bars 100 pm. FIG. 2D shows quantification of neuronal c-Fos expression in the anti-CSF1 R Ab group (n = 10 mice) relative to the control Ab group (n = 7 mice). FIG. 2E is an experimental strategy for local depletion of hippocampus microglia by anti-CSF1 R Ab injection. FIG. 2F shows discrimination index in the NOR test for the mice with intrahippocampal injections of control Ab (n = 5 mice) and anti-CSF1 R Ab (n = 6 mice). FIG. 2G contains representative images of TSPO expression in I ba1 + microglia of theTH Docket No. 222120-2130 thalamus and hippocampus (HPC) 21 days after cortical brain injury. Scale bar, 10 pm. FIG. 2H shows quantification of microglial TSPO expression in HPC relative to thalamus (n = 3 mice per region). FIG. 2I is an experimental timeline for DREADD experiments. Tmem119 CreERT2hM3Dq-mCitrinel_SL / LSL mice received intraperitoneal (i.p.) injections of tamoxifen (TAM) injections for five consecutive days to induce hM3D(Gq)-mCitrine expression in Tmem119+ microglia. Then the mice were implanted with cannula into the thalamus. Two weeks after the cannula implantation, the mice underwent the NOR test. CNO or vehicle (Veh) were injected at the habituation and training sessions of the NOR test. FIG. 2J contains representative images of HA-tagged hM3D(Gq) DREADD expression in Iba1+ microglia in the thalamus in Tmem119 CreERT2hM3Dq-mCitrinel_SL / LSL mice after TAM injections and CNO or vehicle (Veh) injection. FIG. 2K shows discrimination index in the NOR test for vehicle (Veh)- (n = 7 mice) and CNO-treated group (n = 10 mice). Data are shown as the mean ± s.e.m. *p < 0.05, **p < 0.01 ; n.s., not significant; Student’s t-test.

[0010] FIGs. 3A to 3G shows microglia with high expression of Cd9 and phagocytosis- related genes increase in the thalamus after cortical injury.

[0011] FIG. 3A shows uniform manifold approximation and projection (UMAP) plot highlighting 6 microglia subclusters after cortical brain injuries. FIG. 3B contain dot plots showing categorization of each cluster based on gene expression features. Absolute NES was expressed by size of circle, and positive and negative were shown by their color. The gene set enrichment analysis (GSEA) was performed using gene sets representing each microglial state. Multiple comparisons were corrected with the Benjamini-Hochberg method, with all the displayed data showing adjusted p-values below 0.05. FIG. 3C contains feature plots showing P2ry12, Tmem119, Cd9, Lpl, Ifitm3, and Statl expression in microglial cell clusters. FIG. 3D shows pseudotime analysis showing a cell transition trajectory from MG#2 / 3 to MG#4 or MG#6 via MG#1. FIG. 3E contains bar plots showing the ratio in each cluster of the thalamus and the hippocampus. FIG. 3F is a volcano plot of differentially expressed genes in the thalamus compared to the hippocampus among MG#4. FIG. 3G shows spatial distribution of MG#1 , MG#2 / 3, MG#4, and MG#6 in the thalamus 21 days after cortical injuries. Brain areas predominantly occupied by microglia with distinct states are shown with respective colors: MG#1 , MG#2 / 3, MG#4, and MG#6. Representative images of MG#1 (Tmem119lo CD9lo Iba1+), MG#2 / 3 (Tmem119hi Iba1+), MG#4 (CD9hi Iba1+), and MG#6 (Ifitm3hi Iba1+) are shown in each thalamic subregion: paravetricular nucleus (PV), central medial nucleus (CM), nucleus reunions (Re), dorsal lateral geniculate nucleus (DLG), posterior nucleus (Po), ventral posteomedial nucleus (VPM), ventral posteorateral nucleus (VPL), reticular nucleus (Rt), zonaTH Docket No. 222120-2130 incerta (Zl), paracentral nucleus (PC), ventromedial nucleus (VM), and lateral posterior nucleus (LP). Scale bar; 10 pm.

[0012] FIGs. 4A to 4H show CD9hi thalamic microglia eliminate synapses and cause recognition memory deficits in the cortically injured mice.

[0013] FIG. 4A is an experimental timeline for blocking CD9 or Fey receptor III (FcyRIII) by anti-CD9 Ab or anti-FcyRI 11 Ab injection. FIG. 4B contains representative images of CD68 expression in Iba1+ microglia in the thalamus in control Ab- and anti-CD9 Ab-injected group after cortical brain injuries. Scale bar, 100 pm. Quantification data of average CD68+ area per microglia are shown in bar graphs for control Ab- (n= 9 mice) and anti-CD9 Ab-injected group (n = 10 mice). FIG. 4C contains representative images of PSD95+ signals overlapped with CD68+ lysosomal area of I ba1 + microglia in the thalamus of control Ab- and anti-CD9 Ab-injected mice after cortical brain injuries. Scale bar, 10 pm. PSD95+ signals colocalized to CD68+ lysosomal area (CD68+ PSD95+ area per microglia) are calculated per animal and shown in bar graphs for control Ab- (n= 9 mice) and anti-CD9 Ab-injected group (n = 10 mice). FIG. 4D contains representative images of PSD95+ synaptic puncta of the thalamus in control Ab- and anti-CD9 Ab-injected mice after cortical brain injuries. Scale bar, 10 pm. Quantification data are shown in bar graphs for control Ab- (n = 9 mice) and anti-CD9 Ab-injected group (n = 10 mice). FIG. 4E contains representative images of NeuN+ cells of the thalamus in control Ab- and anti-CD9 Ab- injected mice after cortical brain injuries. Scale bar, 10 pm. Quantification data are shown in bar graphs for control Ab- (n = 9 mice) and anti-CD9 Ab-injected group (n = 10 mice). FIG. 4F contains representative images of neuronal c-Fos expression in the thalamus in control Ab- and anti-CD9 Ab-injected mice after cortical brain injuries. Scale bars 100 pm. Quantification data of neuronal c-Fos expression in the anti-CD9 Ab group (n = 10 mice) relative to the control Ab group (n = 9 mice) are shown in bar graphs. FIG. 4G contains representative images of p-Syk signals in Iba1+ microglia of the thalamus in control Ab- and anti-CD9 Ab-injected group after cortical brain injuries. Scale bar, 10 pm. Quantification data of the p-Syk levels in Iba1 + microglia in the anti-CD9 Ab group (n = 10 mice) relative to control Ab group (n = 9 mice) are shown in bar graphs. FIG. 4H shows discrimination index in the NOR test for control Ab (n - 9 mice) and anti-CD9 Ab group (n = 10 mice). Data are shown as the mean t s.e.m. **p < 0.01, ***p < 0.005; Student’s t-test.

[0014] FIGs. 5A to 5H show extravasated IgGs induce the generation of CD9hi thalamic microglia that eliminate synapses and impair recognition memory in the cortically injured mice. FIG. 5A contains representative images of IgG and CD31+ blood vessels of the thalamus and the hippocampus (HPC) after cortical brain injuries. Percentages of extravascular IgG coverageTH Docket No. 222120-2130 area per total IgG coverage area are shown in bar graphs for the injury (n = 4 mice) and the sham (n = 4 mice) group. Scale bar, 10 pm. FIG. 5B contains representative images of CD9 and FcyRIH expression in the same Iba1+ microglia. Scale bar, 10 pm. FIG. 5C contains representative images of IgG and CD31+ blood vessels in the thalamus of cortically injured mice treated with control Ab and anti-CD9 Ab. Scale bar, 100 pm. Percentages of extravascular IgG coverage area per total IgG coverage area are quantified for the control Ab (n = 10 mice) and the anti-CD9 Ab (n = 9 mice) group. FIG. 5D contains representative images of CD9hi Iba1+ thalamic microglia in control Ab and anti-FcyRI 11 Ab injected mice after cortical brain injuries. Quantification data are shown in bar graphs for control Ab (n = 10 mice) and anti- FcyRIH Ab group (n = 9 mice). Scale bar, 10 pm. FIG. 5E contains representative images of lgG+ signals overlapped with CD68+ lysosomal area in Iba1+ microglia of the thalamus in control Ab and anti-FcyRI 11 Ab injected mice after cortical brain injuries. White arrow heads show CD68+lgG+ overlaps in microglia. Scale bar, 10 pm. Quantification of average area of CD68+lgG+ overlaps in microglia are shown in bar graphs for control Ab (n= 10 mice) and anti- FcyRIH Ab (n = 9 mice) group. FIG. 5F contains representative images of PSD95+ signals overlapped with CD68+ lysosomal area in Iba1+ microglia of the thalamus in control Ab and anti-FcyRI 11 Ab injected mice after cortical brain injuries. Scale bar, 10 pm. CD68+ PSD95+ areas per microglia are calculated per animal and shown in bar graphs for control Ab (n= 10 mice) and anti-FcyRI 11 Ab group (n = 9 mice). FIG. 5G shows discrimination index in the NOR test for control Ab (n = 10 mice) and anti-FcyRI 11 Ab (n = 9 mice) group. FIG. 5H is a graphic illustration showing how CD9+ microglial state develops in response to extravasated IgG via FcyRIH signaling, engulfing synapses and impairing neuronal function. Data are shown as the mean ± s.e.m. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; n.s., not significant; Student’s t-test.

[0015] FIGs. 6A to 6I show cortical brain injuries induce delayed microglia activation. FIG. 6A contains representative images of the gross brain morphology 21 days after the cortical ablation injury (CAI) and sham surgery. FIG. 6B contains representative images of I ba1 + microglia 21 days after CAI and sham surgery. Scale bars, 1 mm (in low- magnification image) and 100 pm (in high-magnification image). FIG. 6C contains representative images of Iba1 + microglia in the intact brain and the brains on day 7, 14, and 21 post-CAI. Scale bar, 500 pm. FIG. 6D shows delayed secondary microglia activation across various brain regions. Severity of microglia activation is shown as follows: -, no activation; ±, faint activation; +, mild activation; ++, moderate activation; and +++, massive activation. mPFC, medial prefrontal cortex; CC, corpus callosum; PRh, perirhinal cortex; HPC, hippocampus; AD, anterodorsal nucleus; AV, anteroventral nucleus; MD, mediodorsal nucleus; MV, medioventral nucleus; CM, central medialTH Docket No. 222120-2130 nucleus; VM, ventral medial nucleus; VL, ventral lateral nucleus; Po, posterior nucleus; VPL, ventral posterolateral nucleus; VPM, Ventral posteromedial nucleus. FIG. 6E contains representative images of the gross brain morphology 21 days after controlled cortical impact (CCI) and sham surgery. FIG. 6F contains representative images of I ba1 + microglia 21 days after CCI and sham surgery. Scale bars, 1 mm (in low- magnification image) and 100 pm (in high-magnification image). FIG. 6G contains representative images of Iba1+ microglia 21 days after repeated closed CCI and sham surgery. Scale bars, 1 mm (in low- magnification image) and 100 pm (in high-magnification image). FIG. 6H contains representative images of PSD95+ synaptic proteins of the thalamus after cortical brain injuries and sham surgery. Scale bar, 10 pm. Quantification of number of PSD95+ puncta in the thalamus after cortical brain injuries and sham surgery, (n = 4 mice for sham; n = 4 mice for injury). FIG. 6I contains representative entative images of CD68+PSD95+ area in I ba 1 + microglia of the thalamus after cortical brain injuries and sham surgery. Scale bar, 10 pm. Quantification of average area of CD68+PSD95+ signals are shown in bar graphs for the sham (n = 4 mice) and the injury (n = 4 mice) group. Data are shown as the mean ± s.e.m. ***p < 0.001 ; Student’s t-test.

[0016] FIGs. 7 A and 7B show thalamic microglia activation and the NOR deficits in male mice 21 days after cortical injuries. FIG. 7A contains representative images of I ba1 + microglia in the thalamus 21 days after injuries and sham surgery. Scale bar, 100 pm. Quantification of I ba 1 + area is shown (n = 3 per group). FIG. 7B shows discrimination index in the NOR test for sham (n = 5) and injury (n = 8) group. Data are shown as the mean ± s.e.m. **p < 0.01 ; Student’s t-test.

[0017] FIGs. 8A to 8D show chemogenetic activation of thalamic neurons ameliorate the NOR deficits after cortical injuries. FIG. 8A contains representative images of neruonal c-Fos expression in the thalamus, PRh, mPFC, and hippocampus (CA3 and CA1) upon CNO or vehicle administration. FIG. 8B contains representative images of Iba1+ microglia in AAV- injected areas (indicated by mCherry expression) in the thalamus of mice with CNO or vehicle administration. Quantification of Iba1 coverage area is shown on the right (n = 11 for AAV with vehicle; n = 8 for AAV with CNO). FIG. 8C shows effects of CNO injection on thalamic microglia and neurons in injured mice without AAV injection. Upper panel: representative images of Iba1 + microglia in the thalamus after CNO or vehicle administration. Quantification of I ba 1 coverage area is shown on the right (n = 3 per group). Lower panel: representative images of neuronal c- Fos expression in the thalamus after CNO or vehicle administration. Quantification is shown on the right (n = 3 per group). Scale bars, 100 pm. FIG. 8D shows discrimination index in the NORTH Docket No. 222120-2130 test for vehicle and CNO-treated injured mice (n = 3 per group). Data are shown as the mean ± s.e.m. n.s., not significant; Student’s t-test.

[0018] FIGs. 9A to 9F show effects of anti-CSF1 R Ab injection on thalamic and hippocampal microglial activation and recognition memory deficits after cortical injuries. FIG. 9A contains representative images of I ba1 + microglia in the thalamus of mice receiving intrathalamic injections of control Ab or anti-CSF1 R Ab after cortical injuries (CAI). Quantification of Iba1 coverage area is shown on the right (n = 7 for control Ab; n = 10 for anti- CSF1R Ab). Scale bar, 100 pm. FIG. 9B contains representative images of NeuN+ neurons in the thalamus of mice receiving intrathalamic injections of control Ab or anti-CSF1 R Ab after cortical injuries. Quantification of the number of NeuN+ cells is shown on the right (n = 7 for control Ab; n = 10 for anti-CSF1 R Ab). Scale bar, 100 pm. FIG. 9C contains representative images of I ba1+ microglia in the thalamus of mice receiving intrathalamic injections of control Ab or anti-CSF1 R Ab after cortical injuries (CCI). Quantification of I ba1 coverage area is shown on the right (n = 5 for control Ab; n = 3 for anti-CSF1 R Ab). Scale bar, 100 pm. FIG. 9D shows discrimination index in the NOR test for mice with intrathalamic injections of control Ab and anti- CSF1R Ab (n =8 per group) after cortical injuries (CCI). FIG. 9E contains representative images of I ba1 + microglia in the hippocampus of mice receiving intrahippocampal injections of control Ab or anti-CSF1 R Ab after cortical injuries (CAI). Quantification of the number of I ba1+ microglia is shown (n = 5 for control Ab; n = 6 for anti-CSF1 R Ab). Scale bar, 100 pm. FIG. 9F contains representative images of Iba1 + microglia and NeuN+ neurons in the hippocampus 21 days after cortical injuries (CAI) and sham surgeries. Quantification data for each region are shown on the right (n = 3 for sham; n = 5 for injury). Scale bars, 100 pm. Data are shown as the mean ± s.e.m. n.s., not significant; *p < 0.05; **p < 0.01 ; Student’s t-test.

[0019] FIGs. 10A to 10D show induction of reactive microglial changes by intra-thalamic LPS injection causes cognitive impairment in non-injured mice. FIG. 10A is an experimental timeline for cannula implantation, intra-thalamic LPS injection and the NOR test. FIG. 10B contains representative images of Iba1+ microglia and NeuN+ neurons in the thalamus 4 days after vehicle or LPS injection. Quantification data of Iba1 coverage area, the number of Iba1 + microglia, and the number of NeuN+ neurons are shown on the right (n = 7 mice for vehicle; n = 9 mice for LPS). Scale bars, 500 pm (top panel) and 10 pm (middle and bottom panels). FIG. 10C shows discrimination index in the NOR test for vehicle-injected group (n = 7 mice) and LPS-injected group (n = 9 mice). FIG. 10D shows neuronal c-Fos expression in the thalamus, PRh, mPFC, and hippocampus 4 days after vehicle or LPS injection. Scale bars, 100 pm. Data are shown as the mean s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001 ; Student’s t-test.TH Docket No. 222120-2130

[0020] FIGs. 11A to 11 D show chemogenetic induction of reactive changes in thalamic microglia causes cognitive impairment in non-injured mice. FIG. 11A contains representative images of TNF-a producing I ba 1 + microglia and NeuN+ neurons in the thalamus after vehicle or CNO injection. Scale bars, 100 pm (left panel) and 10 pm (right panel). FIG. 11 B shows quantification data of % activated microglia, TNF-a producing Iba1+ microglia, and NeuN+ cells (n = 7 for vehicle; n = 10 for CNO). FIG. 11 C contains representative images of neuronal c-Fos expression in the thalamus, PRh, mPFC, and hippocampus (CA3 and CA1) after vehicle or CNO injection. FIG. 11 D shows quantification of neuronal c-Fos expression changes in CNO- treated group relative to vehicle-treated group. Scale bars, 100 pm. Data are shown as the mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001 ; Student’s t-test.

[0021] FIGs. 12A to 12C show microglia with distinct transcriptional states in the thalamus and hippocampus. FIG. 11A is a UMAP plot showing 19 clusters with 6 microglia subclusters in the hippocampus and the thalamus after cortical injury. FIG. 11 B contains representative images of CD9 expression in Iba1+ microglia in the hippocampus and halamus after cortical brain injuries. Scale bar, 10 pm. Quantification of the number of CD9hi Iba1 + microglia in the hippocampus (n = 3) and the thalamus (n = 3) is shown in bar graphs. FIG. 11C contains representative images of Iba1+ microglia of the thalamus after cortical brain injuries that express CD9 and LPL. Scale bar, 10 pm. Data are shown the mean ± s.e.m. ***p < 0.001 ; Student’s t-test.

[0022] FIGs. 13A to 13C show thalamic microglia cause neuronal dysfunction via FcyRIII and CD9-mediated synaptic elimination and neuronal damage. FIG. 13A contains representative images of IgG and CD31+ blood vessels in the thalamus after cortical brain injuries and sham surgery. Percentages of extravascular IgG coverage area per total IgG coverage area are quantified for the injury (n = 4 mice) and the sham (n = 4 mice) group. Scale bar, 10 pm. FIG. 13B contains representative images of FcyRIII expression of Iba1+ microglia after cortical brain injuries of the thalamus in control Ab- and anti-CD9 Ab-injected group. Scale bar, 10 pm. Quantification of the number of FcyRIII expressing Iba1+ microglia are shown in bar graphs for control Ab (n = 10 mice) and anti-CD9 Ab (n = 9 mice) group. FIG. 13C contains representative images of p-Syk signals in Iba1+ microglia of the thalamus in control Ab- and anti-FcyRI 11 Ab-injected group after cortical brain injuries. Scale bar, 10 pm. Quantification data of the p-Syk levels in Iba1+ microglia in anti-FcyRIII Ab group (n = 9 mice) relative to control Ab group (n = 10 mice) are shown in bar graphs. Data are shown as the mean ± s.e.m. n.s., not significant; **p < 0.01 ; Student’s t-test.

[0023] TH Docket No. 222120-2130

[0024] FIGs. 14A to 14D show the effects of anti-CD9 blockade on neuropathology and cognition in 5XFAD mice, a typical mouse model to investigate Alzheimer’s disease-realted amyloid beta pathology and its impact on cognition. FIG. 14A. contains representative images of high-level expression of CD9 in I ba1 + microglia in the HPC of 5XFAD mice compared to littermate controls (12 months of age). FIG. 14B show the experimental schedule of intrathecal injections of anti-mCD9 antibodies (KMC8.8) and isotype control antibodies. FIG. 14C (left) presents A|3 plaques and synapses (PSD95) in the thalamus of 5XFAD mice (6 months of age) after two weeks of antibody injections. FIG. 14C (right) shows its quantification data in bar graphs. FIG. 14D show the behavioral data in the NOR test using discrimination index for isotype control Ab (n = 5 mice) and anti-CD9 Ab group (n = 4 mice). Scale bars, 100 pm. Bar graphs represent mean ± s.e.m. *p < 0.05; Student’s t-test.DETAILED DESCRIPTION

[0025] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0028] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. TheTH Docket No. 222120-2130 citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0029] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0031] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0032] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0033] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0034] The term “brain injury” refers to any and all injury of the brain and can be caused by fracture or penetration of the skull (i.e. a vehicle accident, fall, gunshot wound), a disease process (i.e. neurotoxins, infections, tumors, metabolic abnormalities, etc.) or a closed head injury such as in the case of rapid acceleration or deceleration of the head (i.e. Shaken Baby Syndrome, blast), blunt trauma, concussions, and concussion syndrome.TH Docket No. 222120-2130Uses

[0035] As disclosed herein, CD9 blocking antibodies, and other binding agents, are useful for treating a traumatic brain injury or neurodegenerative disease involving CD9- expressing microglia.

[0036] There are many complications associated with brain injury. For example, "25% of patients with brain contusions or hematomas and ”50% of patients with penetrating head injuries will develop immediate seizures that occur within the first 24 hours of the injury. These immediate seizures increase the risk of early seizures which are defined as seizures occurring within one week after injury. However, these seizures do not seem to be linked to the development of post-traumatic epilepsy (recurrent seizures occurring >1 week after initial trauma).

[0037] Another complication of brain injury is hydrocephalus or post-traumatic ventricular enlargement. This complication occurs when cerebrospinal fluid (CSF) accumulates in the brain resulting in dilation of the cerebral ventricles and an increase in intracranial pressure (ICP). This condition can develop during the acute stage of brain injury or may not appear until later. Treatment includes shunting and draining of CSF as well as treatment for the root cause of the condition.

[0038] Another complication is when CSF leaks occur following tearing of the meningeal layers that cover the brain. This often occurs following skull fracture. A tear between the dura and the arachnoid membranes can cause CSF to leak out of the subarachnoid space into the subdural space. CSF can also leak from the nose and the ear. In addition, tears that allow CSF to leak out of the brain cavity can also allow air and bacteria into the cavity, possibly causing infections such as meningitis. Infections within the intracranial cavity are a dangerous complication of brain injury. They may occur outside of the dura mater, below the dura, below the arachnoid membrane or within the brain itself (abscess). Most of these complications develop within a few weeks of the initial trauma and result from skull fractures or penetrating injuries. Standard treatment involves antibiotics and sometimes surgery to remove the infected tissue. Meningitis may be especially dangerous, with the potential to spread to the rest of the brain and nervous system.

[0039] Any damage to the head or brain generally will cause some degree of damage to the vascular system serving the brain. While the body can repair damage to small blood vessels, damage to larger vessels can result in serious complications. For example, damage to one of the major arteries leading to the brain can cause a stroke, either through bleeding from the artery (hemorrhagic stroke) or through the formation of a clot at the site of injury (thrombusTH Docket No. 222120-2130 or thrombosis), blocking blood flow to the brain (ischemic stroke). Other types of vascular injuries include vasospasm and the formation of aneurysms. The methods of the invention are also suitable for treating stroke and related complications.

[0040] Skull fractures, especially at the base of the skull, can cause cranial nerve injuries that result in compressive cranial neuropathies. All but 3 of the 12 cranial nerves project out from the brainstem to the head and face. The seventh cranial nerve, called the facial nerve, is the most commonly injured cranial nerve in brain injury and damage to it can result in paralysis of facial muscles.

[0041] Pain, especially headache, is commonly a significant complication for conscious patients following brain injury. Serious complications for patients who are unconscious, in a coma or in a vegetative state include bed or pressure sores, recurrent bladder infections, pneumonia and other life-threatening infections, and progressive multiple organ failure.

[0042] Other complications caused by brain injuries include becoming paraplegia or quadriplegia.

[0043] TBI is the signature injury of the Iraq conflict (Okie (2005) NEJM 352(20):2043- 2047; Okie (2006) NEJM 355:2609-2615; Vasterling et al, (2006) JAMA 296:519-529; Taber et al, (2006) J Neuropsychiatry Clin Neurosci 18:141-145; Das et al, (2005) NEJM 353: 633-634). TBI and its association with post-traumatic stress disorder (PTS) are in the news every day with the latest information revealing that 30% of the injured military population returns home from Iraq with TBI.

[0044] Treatment options for brain injury patients can involve surgery, draining of fluids and rehabilitation. Approximately half of severely head-injured patients will need surgery to remove or repair hematomas or contusions. When an injury occurs inside the skull-encased brain, there is no place for swollen tissues to expand and no adjoining tissues to absorb excess fluid. This increased pressure is called intracranial pressure (ICP) and requires draining of fluid to decrease the ICP. In some instances drugs such as mannitol or barbiturates can be used to decrease ICP. The cognitive and communication problems associated with brain injury are best treated as soon after the injury as possible. This early therapy will frequently center on increasing skills of alertness and attention, improving orientation to person, place, time, and situation, and stimulating speech understanding. Longer term rehabilitation may be performed depending upon the needs of the individual.

[0045] To date, no treatment option exists that is able to ameliorate cellular damage following brain injury, provide neuroprotection or induce the growth and development of new brain cells to replace damaged brain cells, any or all of which could help return the injuredTH Docket No. 222120-2130 patient to normal or near normal function. Therefore, it is an object of the instant invention to provide such treatment options for brain injury patients.Anti-CD9 Blocking Agents

[0046] Disclosed herein are methods for treating a traumatic brain injury in a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of a composition comprising an anti-CD9 blocking agent, such as a blocking antibody.

[0047] Anti-CD9 antibody sequences are described in WO2017119811A1, which is incorporated by reference for the teaching of these antibody sequences. In some embodiments, the anti-CD9 antibody can comprise a variable heavy (VH) domain having CDR1 , CDR2 and CDR3 sequences and a variable light (V ) domain having CDR1 , CDR2 and CDR3 sequences. In some embodiments, the CDR1 sequence of the VH domain comprises the amino acid sequence DYAMH (SEQ ID NO: 1 ); the CDR2 sequence of the VH domain comprises the amino acid sequence GISWNSGSIVYADSVKG (SEQ ID NO:2); the CDR3 sequence of the H domain comprises the amino acid sequence AVSGYYPYFDY (SEQ ID NO:3); the CDR1 sequence of the VLcomprises the amino acid sequence KSSQSVLYSSNNKNYLG (SEQ ID NO:4); the CDR2 sequence of the VL domain comprises the amino acid sequence WASTRES (SEQ ID NO:5); and the CDR3 sequence of the VL domain comprises the amino acid sequence QQYYTTP (SEQ ID NO:6).

[0048] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e. , an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0049] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions,TH Docket No. 222120-2130 substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0050] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic protein of the present disclosure. Methods for the production of singlechain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0051] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VHand two V regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0052] Bivalent and bispecific antibodies can be constructed using only antibody variable domains. A fairly efficient and relatively simple method is to make the linker sequence between the VHand VLdomains so short that they cannot fold over and bind one another. Reduction of the linker length to 3-12 residues prevents the monomeric configuration of the scFv molecule and favors intermolecular VH-VL pairings with formation of a 60 kDa non- covalent scFv dimer “diabody”. The diabody format can also be used for generation of recombinant bis-pecific antibodies, which are obtained by the noncovalent association of two single-chain fusion products, consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody. Reducing the linker length still further below three residues can result in the formation of trimers (“triabody”, about 90 kDa) or tetramers (“tetrabody”, about 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson, 2005, Nature Biotechnology, 23:1126-1136. All of suchTH Docket No. 222120-2130 engineered antibodies may be used in the fusion polypeptides provided herein. Tetravalent Tandab® may be prepared substantially as described in WO 1999057150 A3 or US20060233787, which are incorporated by reference for the teaching of methods of making Tandab® molecules.

[0053] The antigen recognition sites or entire variable regions of the engineered antibodies may be derived from one or more parental antibodies directed against any antigen of interest (e.g., CD30). The parental antibodies can include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using sequences of antibodies or antibody fragments known to be specific for an antigen of interest. Sequences that may be derived from parental antibodies include heavy and / or light chain variable regions and / or CDRs, framework regions or other portions thereof.

[0054] Multivalent, multispecific antibodies may contain a heavy chain comprising two or more variable regions and / or a light chain comprising one or more variable regions wherein at least two of the variable regions recognize different epitopes on the same antigen.

[0055] Candidate engineered antibodies for inclusion in the fusion polypeptides, or the fusion polypeptides themselves, may be screened for activity using a variety of known assays. For example, screening assays to determine binding specificity are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y., 1988, Chapter s.Pharmaceutical composition

[0056] Also disclosed is a pharmaceutical composition comprising a disclosed molecule in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharmaceutical ly-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutical ly-acceptable carrier include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, whichTH Docket No. 222120-2130 matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0057] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0058] Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0059] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.Methods of Treatment

[0060] Also disclosed is a method for treating a traumatic brain injury in a subject by administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition. The disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), byTH Docket No. 222120-2130 intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.

[0061] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0062] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder is affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical daily dosage of the disclosed composition used alone might range from about 1 pg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

[0063] In some embodiments, the molecule is administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1 pg to about 100 mg per kg of body weight, from about 1 pg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of molecule containing lenalidomide administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 pg,TH Docket No. 222120-213010 pg, 100 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.

[0064] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLESExample 1 : CD9hi thalamic microglia drive cognitive impairment after cortical brain injury

[0065] Here, cortical brain injuries in mice were investigated showing that reactive microglia with high expression of CD9 (CD9himicroglia) in the thalamus caused recognition memory deficits via pathological phagocytosis in response to extravasated IgG after cortical brain injuries.ResultsCortical brain injuries induce delayed thalamic microglia changes that correlate with cognitive impairment.

[0066] To identify the reactive microglial changes critical for cognitive impairment after cortical injuries, we first systematically assessed microglial changes in histology across various brain regions after cortical ablation injury targeting primary sensory and motor cortex (Natale, et al. Neuroscience 2002 112:665-676; Omoto, et al. Neurosci Res 2011 69:187-195; Mattugini, et al. Neuron 2019 103: 1086-1095. e1085) (Figs. 6A, 6B). Increased levels of Iba1 protein expression, which associates with reactive microglial changes, were observed in the primary cortical lesion area immediately after injury and gradually attenuated by day 21 (Figs. 1A-1B, 6C-6D). In contrast, I ba1 expression in other brain regions began to increase around 7 days after injury. On day 21 post-injury, the thalamus stood out as the region with the most remarkable Iba1 expression (Figs. 1A-1 B, 6B-6D). The increased Iba1 expression was also associated with microglial morphological changes into round soma with shortened processes (Figs. 1 C, 6B). In the thalamus, microglia changes began in the ventrolateral nucleus (VL) around 7 days after injuries and spread to the anterodorsal nucleus (AD), anteroventral nucleus (AV), centromedian nucleus (CM), ventral posterolateral nucleus (VPL), and mediodorsal nucleus (MD) by day 21 (Fig. 6D). There were also similar microglial reactive changes in other mouse cortical brain injury models, open controlled cortical impact (CCI) and repeated closed CCI (Figs. 6E-6G). In addition to the drastic morphological changes, thalamic microgliaTH Docket No. 222120-2130 expressed high levels of a pro-inflammatory cytokine, tumor necrosis factor-a (TNF-a), and a lysosome-associated protein, CD68 (Figs. 1C-1D). Density of PSD95, a postsynaptic scaffold protein, in the thalamus significantly decreased in mice with cortical injuries compared to sham mice (Fig. 6H). Moreover, PSD95 signals associated with microglial CD68+lysosomes increased in the thalamic microglia (Fig. 6I). These findings suggest that thalamic microglia engulf synaptic components in the thalamus of mice with cortical injuries.

[0067] Mice with cortical injuries also showed deficits in the novel objective recognition (NOR) test on day 21 post-injury (Figs. 1 E-1 F). Notably, a strong correlation was observed between NOR deficits and thalamic Iba1 staining intensity (Fig. 1G). The data are reminiscent of previous findings in human PET imaging data where inflammatory signals related to microglia in the thalamus were correlated with cognitive impairment in TBI patients in a chronic phase of injuries (Ramlackhansingh, et al. Ann Neurol 2011 70:374-383). Although we primarily used female mice in this study, reactive microglial changes and NOR deficits after cortical injuries were similarly observed in male mice (Figs. 7A-7B). Collectively, these data indicate that thalamic microglia changes may be a critical step leading to cognitive impairment after cortical brain injuries.Thalamic neuronal dysfunction triggers cognitive impairment after cortical injuries.

[0068] NOR is regulated by a network of neurons in multiple brain regions, including the perirhinal cortex (PRh), medial prefrontal cortex (mPFC), and hippocampus (HPC), in addition to the thalamus (Hales, et al. Learn Mem 2015 22:83-91 ; Warburton, et al. Behav Brain Res 2015 285:131-139). In the mice with cortical injuries, a mild but significant neuronal loss, in addition to synaptic changes (Fig. 6H), was observed in the thalamus on day 21 post-injury (Figs. 1 H, 11). Reduced expression of neuronal c-Fos proteins, an indicator of neuronal activities, after the NOR test indicated impaired neuronal activation in the thalamus (Figs. 1J, 1 K). Notably, c-Fos expression changes were also observed in the PRh, mPFC, and HPC. These results implied that thalamic neuronal dysfunction caused recognition memory deficits by negatively impacting the neuronal activities linked to NOR. To address this possibility, thalamic neuronal activities were locally enhanced using a chemogenetic approach with the Designer Receptors Exclusively Activated by Designer Drug (DREADD) receptor in the injured mice (Fig. 1 L) (Roth, et al. Neuron 2016 89:683-694). Adeno-associated virus vector expressing an activator DREADD [AAV-hSyn-hM3D(Gq)-mCherry] was injected into the thalamus of the mice that received cortical injuries. Then, clozapine-N-oxide (CNO) was administered intraperitoneally to activate DREADD. As expected, neuronal c-Fos expression increased in the thalamus of injured mice (Figs. 1M, 8A). In addition, neuronal c-Fos expression also increased in the PRh, mPFC, andTH Docket No. 222120-2130HPC, supporting our hypothesis that reduced thalamic neuronal activities negatively impact neuronal activities in these brain regions. Under this condition, the CNO-treated injured mice showed an improved NOR (Fig. 1 N). CNO injection alone did not affect microglia activation, neuronal c-Fos expression in these brain areas, or NOR in the injured mice (Figs. 8B-8D). Thus, our data demonstrated that the reduced neuronal activities in the thalamus after cortical injuries triggered NOR deficits.Microglial changes in the thalamus cause neuronal dysfunction and cognitive impairment after cortical injuries.

[0069] To address the primary role of thalamic microglia in neuronal dysfunction and cognitive impairment, anti-colony stimulating factor 1 receptor antibodies were locally injected (anti-CSF1 R Ab), which has been used to deplete macrophage and microglia (Gordon, et al. Nature 2017 545:495-499; Hoeffel, et al. Immunity 201542:665-678; MacDonald, et al. Blood 2010 116:3955-3963; Squarzoni, et al. Cell Rep 2014 8:1271-1279), into the thalamus of injured mice (Fig. 2A). Intra-thalamic anti-CSF1 R Ab injection reduced the number of reactive microglia in the thalamus and attenuated neuronal loss (Figs. 9A-9B). Under this condition, NOR and neuronal c-Fos expression were restored in the injured mice (Figs. 2B-2D). Intra-thalamic anti- CSF1R Ab injection also showed a similar improvement in microglia changes and NOR after CCI (Figs. 9C-9D). Hippocampal microglia have been widely investigated in their connection to cognitive impairment after cortical brain injuries (Henry, et al. J Neurosci 2020 40:2960-2974; Willis, et al. Cell 2020 180 : 833-846. e816). Unexpectedly, local injection of anti-CSF1 R Ab into the hippocampus of injured mice did not restore NOR (Figs. 2E-2F). Indeed, microglial changes in the hippocampus was more subtle than those in the thalamus, with little expression of translocator protein (TSPO), a mitochondrial activation marker and an established target of microglia tracer in human PET imaging (Ramlackhansingh, et al. Ann Neurol 2011 70:374-383; Chen, et al. Pharmacol Ther 2008 118:1-17; Wolf, et al. Nat Commun 2020 11 :2709; Coughlin, et al. JAMA Neurol 2017 74:67-74) (Figs. 2G-2H, 9F). Neuronal loss was also not evident in the hippocampus (Fig. 9F). These data showed that reactive microglial changes after cortical brain injuries varied arcoss brain regions and thalamic microglial changes were required for injury- induced NOR deficits.

[0070] Next tested was whether the reactive changes of thalamic microglia were sufficient to impair NOR. Lipopolysaccharides (LPS) was first injected locally into the thalamus of non-injured mice to induce microglial reactive changes (Fig. 10A). LPS injection resulted in remarkable thalamic microglial reactive changes and NOR deficits, accompanied by reduced neuronal c-Fos expression in the thalamus and other NOR-related brain regions (Figs. 10B-TH Docket No. 222120-213010D). A recently reported method of chemogenetic activation of microglia was used (Binning, et al. Brain Behav Immun 2020 88:791-801). A conditional transgenic mouse line was generated in which an activator DREADD was expressed in microglia (Tmem119CreERT2hM3Dq-mCitrineLSL / LSLmice) and induced thalamic microglial reactive changes by local CNO injection into the thalamus (Fig. 2I). DREADD activation caused reactive microglial changes locally in the thalamus, and impaired NOR and neuronal c-Fos expression (Figs. 2J, 2K, 11A to 11 D). These data demonstrated that reactive microglial changes in the thalamus was sufficient to cause NOR deficits and related neuronal dysfunction.A distinct microglial state with high expression of Cd9 and phagocytosis-related genes is enriched in the thalamus of injured mice.

[0071] To better understand the molecular mechanisms underlying thalamic microglial changes in the injured mice, single-cell RNA sequencing (scRNA-seq) of CD45+cells in the thalamus and hippocampus of injured mice was performed. Nineteen transcriptionally distinct cell types and states were identified, with 6 distinct states belonging to microglia (designated as MG#1-6) (Figs. 3A, 12A; Table 1). These 6 distinct microglial states were then compared to the previously reporeted microglial states, including homeostatic microglia (HM), disease- associated microglia (DAM), and interferon-responsive microglia (I RM) (Figs. 3B; Table 2) (Paolicelli, et al. Neuron 2022 110:3458-3483). MG#2 and 3 were enriched with HM-related genes, such as P2ry12 and Tmem119. MG#4 was enriched with DAM-related genes, such as Cd9 and Lpl. MG#6 was enriched with IRM-related genes, such as Ifitm3 and Statl (Fig. 3C). MG#1 had an intermediate state partially overlapped with HM, DAM, and IRM. MG#5 was different from the other states, showing a significantly reduced expression of ribosomal protein genes, such as Rps16, Rps23, and Rpl19. Trajectory analysis revealed and cofirmed that MG#2 and 3 progressed toward MG#4 or MG#6 through several common intermediate states in MG#1 (Fig. 3D).TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130TH Docket No. 222120-2130

[0072] Notably, the percentages of MG#4 were more than three-fold higher in the thalamus than in the hippocampus while the percentages of other MG states were smilar or moderately increased / decreased between the two tissues (Fig. 3E). Examination of differentially expressed genes also revealed that the genes related to phagocytosis (Spp1, Cd9, and Lpl) and lysosomes (Cfs 7, Ctsb, and Ctsd) were signficantly higher in the thalamus MG#4 than the hippocampus MG#4 (Fig. 3F). Thalamic MG#4 also had a higher expression of CD9 proteins than the hippocampus MG#4 (Fig. 12B). Thus, even among the same MG#4 state, there were signicant increase in the expression of phagocytosis- and lysosome-related genes in the thalamus compared to the hippocampus.

[0073] The spatial distribution of microglia was next examined with distinct states in the thalamus of injured mice by immunohistochemistry (Figs. 3G, 12C). MG#2 and 3, visualized as Tmem119hiIba1+cells (Fig. 3G), were found in a region distant from the center of accumulation of microglia, such as paraventricular nucleus (PV), central medial nucleus (CM), nucleus reuniens (Re), and dorsal lateral geniculate nucleus (DLG). MG#4, visualized as CD9hiIba1+cells (or LplhiI ba1+cells) (Fig. 3G), were distributed around posterior nucleus (Po), ventral posteromedial nucleus (VPM), and ventral posterolateral nucleus (VPL), regions where microglial morphological changes were most remarkable. MG#6, visualized as Ifitm3hilba1+ cells (Fig. 3G), was found in reticular nucleus (Rt) and zona incerta (Zl). MG#1 , visualized as cells expressing Tmeml 19 and CD9 at lower levels (Fig. 3G), was observed in paracentral nucleus (PC), ventromedial nucleus (VM), and lateral posterior nucleus (LP). In summary, MG#4 (CD9hi) was highly accumulated at the area where remarkable microglial morphologicalTH Docket No. 222120-2130 changes and the highest expression of I ba1 proteins were observed (blue area), MG#6 (Iftm3hi) was located at the periphery, MG#2 and 3 (Tmem119hi) located at distant sites, and MG#1 (Tmeml 19l0CD9l0) was located between the areas occupied by MG#4 and #2 / 3 (Fig. 3G). The localization of MG#4 in the area where microglia showed the most remarkable morphological changes indicated their pivotal role in thalamic neuroinflammation and neuronal dysfunction after cortical injury.CD9hithalamic microglia eliminate synapses and impair recognition memory in the injured mice.

[0074] As CD9 was shown to be involved in macrophage phagocytosis (Kaji, et al. J Immunol 2001 166:3256-3265), it was hypothesized that CD9 signaling drove microglial phagocytosis of synaptic components in the thalamus of injured mice, leading to impaired recognition memory. Previous studies successfully used anti-CD9 blocking Ab to inhibit CD9 function in phagocytosis and other biological processes under in vitro and in vivo conditions (Cho, et al. Cell Death Differ 2020 27:2681-2696; Noda, et al. Int Immunol 201325:643-650; Sangsri, et al. Sci Rep 2020 10:17972). Therefore, anti-CD9 blocking Ab was locally injected into the thalamus via cannula to test if microglial phagocytosis and other thalamic neuropathological changes are attenuated (Fig. 4A). Multiple anti-CD9 Ab local injections from post-injury day 7 to 19 reduced the area covered by CD68+lysosomes per microglia and limited the amount of PSD95 in CD68+area per microglia on post-injury day 21 (Figs. 4B-4C), indicating the attenuated microglial engulfment of synapses. Consistently, anti-CD9 Ab injections significantly increased PSD95+ synaptic puncta number, neuronal cell number, and neuronal c-Fos expression in the thalamus of injured mice (Figs. 4D-4F). Mechanistically, CD9 blockade reduced the phosphorylation of spleen tyrosine kinase (Syk) (Fig. 4G), a downstream kinase of CD9 signaling (Keely, et al. Biol Chem 1996271 :26668-26676) and a critical regulator of microglial phagocytosis of amyloid 3 (Ennerfelt, et al. Cell 2022 185:4135- 4152.e4122). Thalamic CD9 blockade also attenuated NOR deficits in the mice with cortical injuries (Fig. 4H). These results suggested that CD9 enhanced thalamic microglial phagocytosis of synapses via Syk phosphorylation, reducing neuronal activities and impairing recognition memory after cortical injuries.Extravasated IgG via Fey receptor III signaling facilitates the generation of CD9hithalamic microglia.

[0075] Extravasated y-immunoglobulins (IgG) significantly accumulated in the thalamus but not in the hippocampus of injured mice (Figs. 5A, 13A) As CD9 was reported to be functionally associated with (FcyRIII) signaling in macrophages (Kaji, et al. J Immunol 2001TH Docket No. 222120-2130166:3256-3265), it was reasoned that anti-CD9 Ab inhibited microglial IgG uptake via FcyRIII and increased the amount of extravasated IgG in the thalamus. Indeed, the expression of FcyRIII in CD9hithalamic microglia was detected (Fig. 5B). Anti-CD9 Ab injections, however, did not change the levels of extravasated IgG or microglial FcyRIII expression (Figs. 5C, 13B). In contrast, anti-FcyRI II blocking Ab injections reduced CD9 expression and Syk phosphorylation in the thalamic microglia (Figs. 5D, 13C). Anti-FcyRI II Ab injections also reduced microglial phagocytosis of extravasated IgG and synaptic component PSD95 (Figs. 5E-5F) and attenuated NOR deficits in the injured mice (Fig. 5G). These findings suggest that extravasated IgG in the thalamus facilitates the generation of CD9hithalamic microglia via FcyRIII signaling in microglia (Fig. 5H).

[0076] The studies on the effects of CD9 blockade were extended on mouse models with Alzheimer’s disase (AD)-relevant amyloid pathologies. It was confirmed that CD9 was highly expressed in the microglia of 5XFAD mice, a typical mouse model to study AD-related amyloidpathology and its impact on cognition, but not in age-matched non-transgenic control mice (FIG. 14A). Notably, the injection of anti-CD9 antibody into the lateral ventricle to block CD9 function throughout the brain parenchyma showed no significant changes in A[3 deposition or microglial phagocytosis of Ap plaque in the hippocampus (FIG. 14B, 14C). Nevertheless, CD9 blockade improved recognition memory in the novel object recognition test (NOR) (FIG. 14D). Further IHC experiments revealed that synaptic loss was attenuated upon CD9 blockade (FIG. 14C). These findings suggest that CD9 blockade improves cognitive function by inhibiting microglial phagocytosis of synapses. The effects of CD9 seem distinct from those of other known DAM-related molecules, such as TREM2 and CLEC7a, the blockade of which enhances cognitive impairment by attenuating microglial phagocytosis of Ap plaque and increasing Ap plaque. Therefore, these data have revealed that CD9 blockade has a unique therapeutic potential on AD-related microglial reactive changes and cognitive impairment, beyond TBI- associated microglial changes and cognitive impairment.Discussion

[0077] The present study demonstrated that reactive microglial changes enriched in the thalamus drive cognitive impairment after cortical injuries in mice. Reactive microglial changes in the thalamus gradually became apparent around 7 days after cortical injuries and reached their peak by 21 days and were qualitatively different from those in the hippocampus. Notably, local microglia depletion in the thalamus, but not in the hippocampus, attenuated injury-induced cognitive impairment. Conversely, local induction of reactive changes in the thalamus by chemogenetics and inflammatory stimuli was sufficient to cause neuronal dysfunction andTH Docket No. 222120-2130 cognitive impairment in non-injured mice. Microglia were further identified with several distinct states in the thalamus and hippocampus of the cortically injured mice. The microglial state characterized by high-level expression of CD9 and phagocytosis-related genes was more enriched in the thalamus than in the hippocampus. Blocking CD9 in the thalamus attenuated recognition memory deficits by reducing phagocytosis of synaptic components and restoring neuronal activities in the thalamus and its projected areas. CD9 expression in microglia was induced by the engagement of FcyRIII by extravasated IgG in the thalamus after brain injuries. FcyRIII blockade in the thalamus also attenuated recognition memory deficits by cortical injuries. These findings provided a novel insight into the mechanisms of how reactive microglial changes in a specific brain region, the thalamus, contribute to cognitive impairment after cortical injuries.

[0078] Chronic inflammatory changes in the thalamus have been observed in multiple studies of human TBI patients (Nordstrom, et al. PLoS Med 2018 15:e1002496; Barnes, et al. JAMA Neurol 2018 75:1055-1061 ; Coughlin, et al. JAMA Neurol 2017 74:67-74). A recent study using a rodent model of cortical brain injuries has also highlighted the thalamic microglial changes in the context of injury-induced sleep spindle loss and epileptic spikes (Willis, et al. Cell 2020 180: 833-846. e816). Nevertheless, it remains unclear whether thalamic microglial changes contribute to cognitive impairment following brain injuries. This study is the first to demonstrate the requirement of thalamic microglia for cortical injury-induced deficits in cognitive impairment. Local microglial depletion using anti-CSF1 R Ab has allowed us to address the contribution of microglia in the thalamus to neuronal dysfunction and cognitive impairment. This Ab-based depletion method can be employed to determine the effects of local and transient depletion of microglia on brain and behavioral outcomes. Thus, it can be advantageous to more popular microglia depletion methods using pharmacological methods (e.g., CSF1 R inhibitors, such as PLX5622) and genetically engineered mice (e.g., mice expressing diphtheria toxin receptor) when one needs to examine the contribution of region-specific microglial changes in a specific timeframe to neuronal function and behavior.

[0079] These local microglia depletion experiments have also revealed that, unlike thalamic microglia, hippocampal microglia did not drive recognition memory deficits after cortical injuries. Moreover, these data show that local induction of reactive microglia in the thalamus by LPS injection or DREADD is sufficient to impair recognition memory. These findings are somewhat unexpected considering the well-established role of the hippocampus in learning and memory but may reflect the differential activation status between thalamic and hippocampal microglia after cortical injuries. Compared to the thalamic microglia, the hippocampal microgliaTH Docket No. 222120-2130 were generally less activated and not associated with the expression of TSPO, a marker for mitochondria activation (Chen, et al. Pharmacol Ther 2008 118:1-17; Wolf, et al. Nat Commun 2020 11 :2709). At the gene expression levels, the microglia with high-level expression of Cd9 and Spp1 were much less in the hippocampus than in the thalamus. It remains unclear what causes this microglial activation difference between the thalamus and hippocampus. One plausible explanation is that damages to the highly dense connections between the thalamus and primary cortical injury areas result in the release of an excessive amount of axonal debris and blood-brain-barrier breakage in the thalamus, enhancing microglial acquisition of phagocytic phenotype (Donat, et al. Front Aging Neurosci 2017 9:208). Another possibility is that microglia in the thalamus may be more prone to be activated than those in other brain areas. Recent studies in rodents and humans support the regional difference in microglial function (Colombo, et al. Nat Neurosci 2022 25:1379-1393; Lopes, et al. Nat Genet 2022 54:4-17). Future studies will address the relative contributions of these extrinsic and intrinsic factors to thalamic microglial changes after cortical injuries.

[0080] The scRNA-seq analysis identified microglia with multiple distinct states, including Tmem119hihomeostatic, CD9hiphagocytic, and Ifitm3hiIFN-responsive microglia in the thalamus of cortically injured mice. These microglia showed distinct spatial localization patterns. Specifically, CD9himicroglia and Ifitm3himicroglia existed in different thalamic subregions in an almost mutually exclusive manner.

[0081] CD9 has been known as a marker for disease-associated microglia (DAM), first described in studies on a mouse model of Alzheimer’s disease (Keren-Shaul, et al. Cell 2017 169:1276-1290. e1217). Multiple subsequent studies suggest that DAM and similar disease- associated microglial states (e.g., MGnD, ARM) may play either neuroprotective or neurotoxic function in various disease conditions (Paolicelli, et al. Neuron 2022 110:3458-3483), but the experimental evidence for their causal roles in neuronal dysfunction and cognitive impairment are lacking. In this study, CD9himicroglia were functionally targeted in the thalamus of the mice with cortical injuries showing that they disturbed neuronal function by eliminating synapses and impaired recognition memory deficits. Another recent study using a spinal cord nerve injury model has shown a neuroprotective role of microglia expressing CD11c, another DAM-related marker, in injury-associated inflammation and neuropathic pain behaviors (Kohno, et al. Science 2022 376:86-90). These emerging studies addressing the causal role of microglia with distinct states in each disease-relevant context will lead to a better understanding of disease-modifying functions by microglia with specific states under chronic inflammation in various neurologicalTH Docket No. 222120-2130 disorders. Notably, CD9 has also been shown to be involved in AD-related synapse loss in the thalamus and recognition memory deficits.

[0082] FcyRIII inhibition reduced the number of CD9himicroglia in the thalamus. FcyRIII inhibition may suppress the transcriptional activation of CD9 expression in downstream of FcyRIII signaling (Bournazos, et al. Nat Rev Immunol 2020 20:633-643). As CD9 and other tetraspanins are known to co-localize with FcyRs (Saiz, et al. Front Immunol 2018 9:1074), the transcriptional induction of CD9 expression by FcyRs may act as a feed-forward mechanism to generate the CD9-FcyRIII complex on the plasma membrane, facilitating the phagocytosis of IgG. Extravasated IgG may aberrantly tag healthy synapses and allow them to be eliminated by FcyR-dependent phagocytosis. Alternatively, CD9 induction may facilitate FcyR-independent phagocytosis mechanisms, such as those mediated by the complement system (Hong, et al. Science 2016 352:712-716). Either way, our study has highlighted that FcyRIII-dependent CD9 induction in microglia plays a critical step in the transition of cortical brain injury into chronic inflammation. Targeting the molecular mechanisms involving FcyRIII-dependent CD9 induction by IgG may provide a potential therapeutic strategy to prevent cognitive impairment after cortical brain injury.Materials and MethodsMice

[0083] C57BL / 6J, Tmeml 19-CreERT2 (Tmem119CreERT2), CAG-lox-Stop-lox-hM3Dq- mCitrine (hM3Dq-mCitrineLSL / LSL), and 5XFAD mice were purchased from the Jackson Laboratory. Tmeml 19CreERT2and hM3Dq+ / +mice were crossed to generate Tmeml 19 creERT2|-l|\ / |3Dq.mQitrineLSL / LSLmjce. All the mice were housed in specific pathogen-free facilities with ad libitum access to food and water under a standard light / dark cycle at the University of Alabama at Birmingham. All experimental procedures were performed under the animal protocols approved by the Institutional Animal Care and Use Committees. Female mice were used for most of the experiments unless stated otherwise.Cortical brain injuries

[0084] Cortical Ablation Injury (CAI): Unilateral lesions of the cortex were induced as described previously (Natale, et al. Neuroscience 2002 112:665-676; Omoto, et al. Neurosci Res 2011 69:187-195; Ross, et al. Neuroscience 1990 35:525-550; Omoto, et al. J Neurosci 2010 30:13045-13052; Lukoyanov, et al. Elife 2021 10). After mice were anesthetized, a 3.0 mm-wide burr hole was made in the skull bone at point midway between the lambda and bregma sutures, and laterally midway between the central suture and temporalis muscle. Cortical ablation (1.5 mm each from bregma to caudal and rostral, 3 mm to the right, andTH Docket No. 222120-21301.0 mm in depth) was performed by aspiration with a pipette. In sham-operated mice, after making burr hole, the skin wound was closed without cortical ablation. Compared to a controlled cortical impact (CCI) model, this injury damages the brain tissue by a surgical ablation (contusion) but does not provide any blow or violent shaking (concussion).

[0085] Controlled Cortical Impact (CCI): Moderate CCI was performed as previously described by (Henry, et al. J Neurosci 2020 40:2960-2974; Willis, et al. Cell 2020 180:833- 846.e816; Osier, et al. Methods Mol Biol 2016 1462:177-192; Berkner, et al. Methods Mol Biol 2016 1462:11-28; Barrett, et al. J Neurosci 2020 40:2357-2370). After mice were anesthetized, a 4.0-mm-wide burr hole was made in the skull bone at a point midway between the lambda and bregma sutures, and laterally midway between the central suture and temporalis muscle. After removal of the scalp, the tip of the 3-mm impactor (Leica) piston was angled and kept perpendicular to the exposed cortical surface. Cortical impact was given with the following parameters: impact speed, 3.5 m / s; deformation depth, 1.0 mm; and duration, 400 ms. Sham mice underwent the same craniotomy without cortical impact.

[0086] Repeated closed CCI: Repeated mild closed CCI was performed as previously described (Maynard, et al. Exp Neurol 2020 327:113207; Lynch, et al. J Cereb Blood Flow Metab 2021 41 :1362-1378). Briefly, a midline incision was made to expose the skull. The tip of the 5-mm impactor (Leica) piston was placed vertically 2mm to the right of the bregma. Cortical impact was given with the following parameters: impact speed, 5.0 m / s; deformation depth, 1.0 mm; and duration, 200 ms. Sham mice underwent the same midline incision without cortical impact. Mice received one injury / day for 3 consecutive days.Cannula implantation

[0087] Cannula implantation and microinjection were done as previously described (Mahler, et al. Nat Neurosci 2014 17:577-585; Stachniak, et al. Neuron 2014 82:797-808; Niwa, et al. J Neurosci 200727:7604-7615; Niwa, et al. Science 2013 339:335-339; Matoba, et al. Sci Rep 2017 7:40397). A 26-gauge guide cannula (Plastics One) was implanted ipsilaterally over the right side of thalamus (-1.80 mm AP, +1.50 mm ML and -3.00 mm DV) or right hippocampus (-1.80 mm AP, +1 .50 mm ML and -1 .50 mm DV). The guide cannula was secured in place with anchoring miniature screws and dental cement. A 32-gauge dummy cannula (Plastics One) was inserted into each cannula to prevent clogging.Local antibody and LPS injection

[0088] Anti-CSFIR Ab, anti-CD9 Ab, anti-FcyRIII Ab, and their respective control antibodies were administered via implanted cannula following the schedule indicated in each. LPS (0.5 pl; 4 pg / pl, Sigma) was also injected via cannula. On the injection day, the dummyTH Docket No. 222120-2130 cannula was replaced with a 33-gauge internal cannula (Plastics One) extending 2 mm below the tip of the guide cannula. Unless otherwise stated, 0.5 pl of antibodies or reagents were injected at a rate of 100 nl / min using a microinfusion pump (Narishige). The internal cannula was left in place for an additional 5 min to achieve a proper diffusion of antibodies or reagents.DREADD experiments

[0089] For neuronal DREADD experiments, AAV injection was conducted as previously described (Kano, et al. Sci Signal 2019 12). Briefly, 1 pl of AAV-hSyn-hM3D(Gq) -mCherry (3.23x1012GC / ml; Addgene, 50474) was stereotactically injected into the thalamus (-1.80 mm AP, +0.50 mm ML, and -3.20 mm DV) of WT mice using a 10 pl Nanofil syringe (WPI) at a rate of 200 nl / min immediately after cortical injuries. CNO (10 mg / kg; Cayman, 16882) or vehicle were administered intraperitoneally (i.p.) 30 min before the test session. For microglial DREADD experiments, Tmem119CreERT2hM3Dq-mCit neLSL / LSLmice were injected with 0.5 pl CNO (0.5 mg / mL; Cayman, 16882) or vehicle at a rate of 100 nl / min via an implanted cannula using a microinfusion pump (Narishige) for 3 consecutive days before the analysis.Antibodies

[0090] For immunohistochemistry, the following antibodies were used: I ba1 (rabbit polyclonal; WAKO; 019-19741 , rabbit polyclonal; Abeam; Ab225260, and mouse monoclonal; Sigma-Aldrich; MABN92) at dilutions of 1 :500, NeuN (mouse monoclonal; Abeam; ab190565 and ab177487) at 1 :500, c-Fos (rabbit monoclonal; Cell Signaling; 2250) at 1 :500, TSPO (rabbit monoclonal; Abeam; ab109497) at 1:100, CD68 (rat monoclonal; Novus biological; NBP2- 33337) at 1 :2,000, TNF-a (mouse monoclonal; Abeam; ab1793) at 1 :100, RFP (rat monoclonal; Chromotek; 5F8) at 1 :500, HA-tag (rabbit monoclonal; Cell Signaling; 3724) at 1 :500, CD31 (armenian hamster monoclonal; DSHB; 2H8-s), CD9 (rabbit polyclonal; Proteintech: 20597-1- AP) at 1 :100, FcyRIII (rabbit polyclonal; Abeam; ab203883) at 1:100, p-Syk (rabbit polyclonal; Cell Signaling; 2710T) at 1 :200, PSD95 (mouse monoclonal; Invitrogen; MA1-046) at 1 :500, LPL (goat polyclonal; R & D; AF7197) at 1 :100, Tmem119 (chicken monoclonal; Synaptic Systems; 400009) at 1 :500, Ifitm3 (rabbit polyclonal; Proteintech; 11714-1-AP) at 1 :100.

[0091] For in vivo administration, the following antibodies were injected with a total volume of 0.5 pl per injection: rat lgG2a isotype control (Bio X Cell; BE0089) at dilution of 3.0 pg / pl, anti-CSF1 R (CD115) (Bio X Cell; BE0213) at dilution of 3.0 pg / pl, normal rat IgG control (BD Pharmingen; 553926) at dilution of 1.0 pg / pl, and anti-CD9 (BD Pharmingen; 553758) at dilution of 1.0 pg / pl, normal rat IgG control (R and D; MAB006) at dilution of 0.5 pg / pl, and anti- FcyRIII (R and D; MAB19601) at dilution of 0.5 pg / pl.TH Docket No. 222120-2130Immunohistochemistry

[0092] Mice were anesthetized and transcardially perfused with ice-cold 0.1 M phosphate-buffered saline (PBS) (pH 7.4) followed by 4% paraformaldehyde (PFA). Brain tissue was post-fixation in 4% PFA overnight, followed by 15% and 30% sucrose. Free-floating sections (30 pm in thickness) were prepared by a Leica cryostat, placed in blocking buffer (5% normal goat serum and 0.1 % Triton X-100 in PBS) for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C. After washing in PBS, the sections were further incubated with 1 :500 dilutions of fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature, followed by DAPI staining (10 pg / ml) for 10 min. The sections were mounted on slide glasses with ProLong Diamond antifade mounting medium (Thermo Fisher Scientific) or Fluorescent mounting media (DAKO). Images were acquired using Zeiss LSM 800 Airyscan confocal microscopes and a ZEN software (Zeiss).Image analysis

[0093] Image analysis was performed as previously described (Kano, et al. Sci Signal 2019 12; Dohi, et al. eNeuro 2017 4; Ayata, et al. Nat Neurosci 201821 :1049-1060). Z-stack images were used for evaluation. Image processing and subsequent quantifications were carried out on Imaged (National Institute of Health). The “Measure Object Area” module was used to measure the I ba1 coverage area. The number of Iba1+, NeuN+, and c-Fos+cells per each visual field was quantified using “Analyze Particles” module. To determine the coverage area of CD68+, p-Syk+, PSD95+and lgG+signals in Iba1+ cells, the boundaries of CD68+, p- Syk+, PSD95+and lgG+areas were traced and then superimposed onto the Iba1+images using Imaged. Then, the sizes of double-positive areas were measured and divided by the number of I ba 1+microglia in the field to obtain the average size of CD68+, p-Syk+, PSD95+, and lgG+areas per Iba1+microglia. Then the data from three to five brain sections per mouse were averaged to represent each mouse. To evaluate the distribution of various microglia across the thalamus, a total of 30 images were acquired from the right thalamus 21 days after cortical injury. The fluorescent intensities of Tmem119, CD9, and Ifitm3 signals in Iba1+microglia were measured in each area. If the Tmeml 19 intensity was the highest, the area was defined as MG#2 / 3-rich area. Similarly, the area with the highest CD9 and Ifitm3 signals were defined as MG#4 and MG#6-rich area. The area with similar intensities for CD9 and Tmeml 19 were defined as MG#1-rich area.Single-cell RNA-seq (scRNA-seq)

[0094] Sample preparation and sequencing. Thalamic and hippocampus tissues from the injured hemispheres were micro-dissected. Cells were harvested following theTH Docket No. 222120-2130 manufacturer’s protocol and as described previously (Herron, et al. STAR Protoc 2022 3:101670). Briefly, tissue was transferred into 2 ml of pre-chilled homogenization buffer [15 mM HEPES buffer, 1 mg / mL DNasel (Roche), and Protector RNase inhibitor at 1 :200 (Millipore)]. Tissues were carefully homogenized 10-12 times with Dounce homogenizers, filtered with 70 pm cell strainers, and centrifuged at 2500 rpm for 2 min to pellet cells. Then, debris were removed using Debris remove solution (Miltenyi) following the manufacture’s instruction. Equal numbers of cells from four individual mice thalamus and hippocampus were pooled, stained with LIVE / DEAD and CD45, and sorted using FACS Aria II at the UAB Comprehensive Flow Cytometry Core (CFCC). Then, scRNA-seq libraries were constructed on the 10x Genomics platform using the Single Cell 3’ protocol (v.3.1 , 10x Genomics). The libraries were sequenced using NovaSeq6000 at the UAB Genomics Core facility.

[0095] Data analysis. Raw scRNA-seq data were processed using the Cell Ranger software (v.6.0.2 10x Genomics) for sample demultiplexing, barcode processing, and single-cell counting. Reads were aligned to the GRCm38 (mm10) mus musculus reference genome. The outputs from the Cell Ranger software analysis were then imported into the Seurat package (v.4.0) in R (v.4.1.1) and the subsequent analysis was conducted with the Seurat. For quality control, cells with mitochondrial content more than 2% and extreme unique feature counts (more than 7,500 and less than 200) were removed. Data were normalized with a scaling factor of 10,000. The data from multiple libraries were then combined using FindlntegrationAnchors() and lntegrateData() functions. Principal component analysis (PCA) was performed using the combined data. A scree plot was generated using ElbowPlot() function to select the principal components (PCs) by localizing the last PC before the explained variance reaches a plateau. Selected PCs were used to calculate nearest-neighbor distances and perform Louvain clustering with FindNeighbors() and FindClusters() functions. Then, uniform manifold approximation and projection (UMAP) was used to visualize clusters.

[0096] Cell types were initially annotated using SingleR, and then refined based on the expression of known marker genes in the genes significantly enriched for each cluster that was obtained using the FindAIIMarkers() function. Wilcoxon rank-sum test was used to identify genes differentially expressed between injury and sham groups per each cluster. A 6-cluster subset of the 21 identified clusters that were annotated as microglia was chosen for further microglia cluster classification. Gene set enrichment analysis (GSEA) was performed for the microglia cluster data using gsea() function of Clusterprofiler (v. 4.8.3) (Wu, et al. Innovation (Camb) 2021 2:100141 ; Yu, et al. Omics 2012 16:284-287). To visualize the GSEA result, DotplotQ and ggplotQ function were employed. Gene sets for homeostatic microglia (HM),TH Docket No. 222120-2130 disease-associated microglia (DAM), and interferon-responsive microglia (I RM) were created according to previous study (Dolan, et al. Nat Immunol 2023 24:1382-1390; Yin, et al. Nat Neurosci 202326:1196-1207; Gulen, et al. Nature 2023620:374-380; Silvin, et al. Immunity 2022 55:1448-1465. e1446). To clarify different cluster composition between thalamus and hippocampus, cell number and normalized ratio in each cluster were counted. The result was visualized using ggplot() function. Gene expression patterns were visualized by Seurat FeaturePlot() function.

[0097] Pseudotime analysis was performed using Monocle 3 on the 6 microglial clusters. These clusters were first randomly downsampled to 1000 cells per cluster, and microglia cluster 3 was set as the pseudotime root cluster based on the expression profile matching that of known homeostatic microglia. Pseudotime was then calculated and plotted for visualization. Additionally, a list of genes identified as important in calculating pseudotime was generated using Monocle 3.

[0098] Distribution of cells that highly express specific genes were visualized by Seurat FeaturePlot() function. Findmarkers() function was used to investigate the differential gene expression between MG#4 in thalamus and MG#4 in hippocampus, then visualized as volcano plot using ggplot() function.Behavioral assays

[0099] The novel object recognition (NOR) test evaluates the tendency of rodents to discriminate between new and familiar objects and is used to evaluate recognition memory in mice (Vogel-Ciernia, et al. Curr Protoc Neurosci 2014 69:31.1-17). The test mice were individually habituated for 10 min for consecutive 2 days in an open field box (45 x 45 centimeters; Harvard Apparatus). On the day of the training session, the test mice explored two objects of the same material and shape placed in a symmetrical position for 10 min in the open field box. In the test session (24 hours after the training session), the test mice were again placed into the same box but one of the objects was replaced with a novel object. The exploratory behaviors of the test mice for 10 min were recorded and analyzed using Ethovision XT17 (Noldus). Exploration was defined as reaching the object, sniffing it within less than 2-cm distance, and / or touching it with the nose. Discrimination index (DI) was calculated as ([time spent at the novel object] - [time spent at the familiar object]) / ([time spent at the novel object] + [time spent at the familiar object]).Statistical analysis

[0100] Data were analyzed with Student’s t test and one-way ANOVA using Microsoft Excel and GraphPad Prism 8 (GraphPad Software) unless stated otherwise. Post hoc analysesTH Docket No. 222120-2130 for one-way ANOVA were performed using Dunnett’s test method. Significant differences were considered at p < 0.05.

[0101] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0102] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

TH Docket No. 222120-2130CLAIMS1. A method for treating a traumatic brain injury in a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a blocking antibody, wherein the blocking antibody comprises an anti-CD9 blocking antibody, an anti-FcyRI II blocking antibody, or a combination thereof.

2. The method of claim 1 , wherein the blocking antibody is an anti-CD9 blocking antibody.

3. The method of claim 1 , wherein the blocking antibody is an anti-FcyRIII blocking antibody.

4. The method of claim 1 , wherein the traumatic brain injury is selected from the group consisting of Shaken Baby Syndrome, blast injury, blunt trauma, concussion, and concussion syndrome.

5. The method of claim 1 , wherein the composition is administered by intrathalamic injection.

6. The method of claim 1 , wherein the composition is administered by intravenous injection.

7. The method of claim 1 , wherein the method is performed once.

8. The method of claim 1 , wherein the method is performed daily for from about 2 days to about 21 days.

9. The method of claim 1 , wherein performing the method inhibits CD9 function in phagocytosis.

10. The method of claim 1 , wherein performing the method reduces CD68 area in Iba1 + microglia in the subject by at least about 50% relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.11 . The method of claim 1 , wherein performing the method attenuates microgial engulfment of synapses in the subject by at least about 25% relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

12. The method of claim 1 , wherein performing the method increases PSD95+ synaptic puncta number by at least about 30%, neuronal cell number by at least about 15%, neuronal c-TH Docket No. 222120-2130Fos expression by at least about 95%, or any combination thereof, in the subject, relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

13. The method of claim 1 , wherein performing the method reduces phosphorylation of spleen tyrosine kinase in the subject by at least about 18%, relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

14. The method of claim 1 , wherein performing the method increases ability to memorize a previous observation and discriminate a novel observation by more than two-fold relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody15. The method of claim 1 , wherein the subject is a mammal.

16. The method of claim 1 , wherein the mammal is a human, non-human primate, dog, cat, cattle, swine, sheep, goat, horse, hamster, guinea pig, rat, or rabbit.

17. A method for treating a neurodegenerative disease involving CD9 expressing microglia in a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a blocking antibody, wherein the blocking antibody comprises an anti-CD9 blocking antibody, an anti-FcyRI 11 blocking antibody, or a combination thereof.

18. The method of claim 17, wherein the blocking antibody is an anti-CD9 blocking antibody.

19. The method of claim 17, wherein the blocking antibody is an anti-FcyRI 11 blocking antibody.

20. The method of claim 17, wherein the neurodegenerative diseases is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, frontotemporal dementia, and amyotrophic lateral sclerosis.

21. The method of claim 17, wherein the composition is administered by intrathalamic injection.

22. The method of claim 17, wherein the composition is administered by intravenous injection.TH Docket No. 222120-213023. The method of claim 17, wherein the method is performed once.

24. The method of claim 17, wherein the method is performed daily for from about 2 days to about 21 days.

25. The method of claim 17, wherein performing the method inhibits CD9 function in phagocytosis.

26. The method of claim 17, wherein performing the method attenuates microgial engulfment of synapses in the subject by at least about 25% relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

27. The method of claim 17, wherein performing the method increases PSD95+ synaptic puncta number by at least about 30%, neuronal cell number by at least about 15%, neuronal c- Fos expression by at least about 95%, or any combination thereof, in the subject, relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

28. The method of claim 17, wherein performing the method reduces phosphorylation of spleen tyrosine kinase by at least about 18% in the subject relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

29. The method of claim 17, wherein performing the method reduces CD68 area in Iba1 + microglia in the subject by at least about 50% relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

30. The method of claim 17, wherein performing the method increases ability to memorize a previous observation and discriminate a novel observation by more than two-fold relative to an otherwise identical subject who has not been administered the composition comprising the blocking antibody.

31. The method of claim 17, wherein the subject is a mammal.

32. The method of claim 17, wherein the mammal is a human, non-human primate, dog, cat, cattle, swine, sheep, goat, horse, hamster, guinea pig, rat, or rabbit.

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