WO2021076964A1 - Stathmin 2 (stmn2) as a therapeutic target for parkinson's disease - Google Patents

Abstract

The present disclosure relates to a method for treating a neurodegenerative disorder characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites in an individual diagnosed with or suspected of having a loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. The method comprises administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2. Also disclosed herein is a method for the treatment of an individual with PD comprising administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2, and a vector comprising a nucleic acid encoding STMN2.

Description

STATHMIN 2 (STMN2) AS A THERAPEUTIC TARGET FOR PARKINSON’S

DISEASE

[0001] This application claims benefit of U.S. Provisional Patent Application Serial No.

62/916,752, filed on October 17, 2019, which is hereby incorporated by reference in its entirety. [0002] This invention was made with government support under NS060809 and

NS094733 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The present application contains a Sequence Listing, created on October 16, 2020; the file, in ASCII format, is designated as 3710045AWO_sequencelisting_ST25.txt and is 14.7 kilobytes in size. The file is hereby incorporated by reference in its entirety into the instant application.

FIELD

[0004] The present disclosure relates generally to Stathmin 2 (STMN2) as a therapeutic target for Parkinson’s Disease.

BACKGROUND

[0005] Parkinson’s disease (“PD”) is a common neurodegenerative disorder characterized pathologically by the loss of dopaminergic (“DAergic”) neurons at the substantia nigra and the presence of Lewy body and Lewy neurites in affected brain regions. However, the pathogenic mechanisms of PD remain largely elusive. Previous research has identified over 20 PD causal mutations. Identification of genetic variants in SNCA, LRRK2, PINK1, DJ-1 and Parkin , etc. has shed light on molecular mechanisms of inherited forms of PD. Hernandez et al., “Genetics in Parkinson Disease: Mendelian Versus Non-Mendelian Inheritance,” Journal of Neurochemistry 139(Suppl l):59-74 (2016). Furthermore, large-scale analysis such as Genome- Wide Association Study (GWAS) (Pankratz et al., “Meta- Analysis of Parkinson’s Disease: Identification of a Novel Locus, RIT2,” Ann. Neurol. 71 :370-384 (2012)) has predicted over 25 PD-associated risk loci in various cohorts (Pihlstrom et al., “Supportive Evidence For 11 Loci From Genome-Wide Association Studies in Parkinson’s Disease,” Neurobiology of Aging 34:1708.e7-13 (2013); Nalls et al., “Imputation of Sequence Variants for Identification of Genetic Risks for Parkinson’s Disease: A Meta- Analysis of Genome-Wide Association Studies,” Lancet 377:641-649 (2011); and Nalls et al., “Large-Scale Meta-Analysis of Genome-Wide Association Data Identifies Six New Risk Loci for Parkinson’s Disease,” Nature Genetics 46:989-993 (2014)). However, even with the increasing number of PD-associated genes or risk factors, translation of the findings into biological understanding has remained a major challenge. In fact, the available knowledge regarding the genetic factors explains little the complexity of the disease pathogenesis. The functional hierarchy in PD related gene regulation and their roles in specific neuronal functions underlying the disease mechanism remain elusive.

[0006] Although genetic studies of PD have provided crucial insight into disease mechanism, genetic mutations or variants only account for 5-10% of PD cases. Thomas et al., “Parkinson’s Disease,” Human Molecular Genetics 16 Spec No. 2, R183-194 (2007). A majority of PD cases are sporadic and the etiology remains largely unclear. Idiopathic PD can be viewed as the outcome of complex interactions between multiple genes and environmental factors. For example, exposure to particular pesticides is associated with increasing risk of PD (Betarbet et al., “Chronic Systemic Pesticide Exposure Reproduces Features of Parkinson’s Disease,” Nature Neuroscience 3:1301-1306 (2000)) while caffeine consumption was found to reduce PD risk (Ross et al., “Association of Coffee and Caffeine Intake With the Risk of Parkinson Disease,” JAMA 283:2674-2679 (2000) and Ascherio et al., “Prospective Study of Caffeine Consumption and Risk of Parkinson’s Disease in Men and Women,” Annals of Neurology 50:56-63 (2001)). Furthermore, aging is the most significant risk factor for PD; how aging inflicts on the onset of PD remains unclear. The above notions present a mighty challenge to the investigation of the common causes behind idiopathic PD and demand the access to more powerful and innovative tools.

[0007] The development of high throughput molecular profiling techniques has shed light on the mechanisms of complex diseases whose pathogenesis is not due to alterations in a single gene or signaling pathway. However, the profiles generated only reflect the ultimate outcome of a disease state with detailed regulatory machinery hidden underneath. Over the last decade, co- expression network analysis has been extensively utilized as an unbiased approach to deconvolute gene coexpression/coexpression patterns in higher organisms to infer potential gene regulation and disease commonality (Bagot et al., “Circuit-Wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility,” Neuron 90:969-983 (2016); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); Zhang et al., “A General Framework For Weighted Gene Co-Expression Network Analysis,” Statistical Applications in Genetics and Molecular Biology 4:Articlel7 (2005); and Zhang et al., “Characterization of Genetic Networks Associated with Alzheimer’s Disease,” Methods in Molecular Biology 1303:459-477 (2016)). A previous study developed an advanced method by integrating genetics, gene expression and clinical traits into multiscale molecular networks for identification of disease-relevant gene modules and key regulators (e.g. TYROBP) in Alzheimer’s disease (“AD”) (Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013)). The network biology study opens a new avenue for the understanding of the complexity of major neurodegenerative diseases; with improving clinical and genomic data, the approach is expected to significantly expand knowledge of causal factors that drive disease onset.

[0008] Unfortunately, such network biology analysis is unavailable for PD as the network biology approach requires a relatively large number of samples while limited means of diagnosis and the absence of readily available biomarkers for PD often lead to missed or erroneous diagnosis (Schrag et al., “How Valid is the Clinical Diagnosis of Parkinson’s Disease in the Community?,” Journal of Neurology, Neurosurgery, and Psychiatry 73:529-534 (2002)). As a result, little is known about the structures of gene interactions and regulations in PD thus hindering understanding of idiopathic PD. What is needed is a method that seeks to establish multiscale gene network models of PD based on an ensemble of all the existing human brain gene expression datasets in PD to reveal global landscape of gene interaction and regulations underlying the pathogenesis of idiopathic PD, and to assist in the development of novel therapeutic targets for PD.

[0009] The present disclosure is directed to overcoming these and other deficiencies in the art.

SUMMARY

[0010] A first aspect of the present disclosure relates to a method for treating a neurodegenerative disorder characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites in an individual diagnosed with or suspected of having a loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. The method includes administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2.

[0011] In one embodiment, the neurodegenerative disorder is Parkinson’s Disease (PD).

In another embodiment, the agent is administered to the central nervous system of the individual. In yet another embodiment, the agent is administered intrathecally, intranasally, intraperitoneally, orally, parenterally, nasally, subcutaneously, intravenously, intramuscularly, intracerebroventricularly, intraparenchymally, by intranasal inhalation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.

[0012] In one embodiment, the agent capable of increasing the expression and/or activity of STMN2 is a vector comprising a nucleic acid encoding STMN2. In another embodiment, the expression vector containing a nucleic acid encoding STMN2 is an adeno-associated viral vector (AAV). In another embodiment, the AAV is AAV9. In another embodiment, the nucleic acid encoding STMN2 has the nucleotide sequence of SEQ ID NO: 5. In one embodiment, Synapsin_Stmn2_IRES__GFP is packaged into AAV9 for STMN2 overexpression in the target tissue.

[0013] A second aspect of the present disclosure relates to a method for the treatment of an individual with PD. The method includes administering to the individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2. [0014] A third aspect relates to a vector comprising a nucleic acid encoding STMN2. In one embodiment, the vector is an adeno-associated viral vector (AAV). In another embodiment, the AAV is AAV9. In one embodiment, the STMN2 is human. In another embodiment, the STMN2 is murine. In yet another embodiment, the human STMN2 comprises the nucleotide sequence of SEQ ID NO: 5, as described herein. In yet another embodiment, the murine STMN2 comprises the nucleotide sequence of SEQ ID NO: 1, as described herein.

[0015] The present disclosure provides a method for treating neurodegenerative disorders like Parkinson’s Disease (PD) based on the use of multiscale transcriptomic network analysis and identification of STMN2 as a regulator of the transcriptomic network underlying PD. The present disclosure presents evidence that Stmn2 deficient mice show dopaminergic neuron vulnerability, phosphorylated α-synuclein elevation, and locomotor function deficits. Furthermore, reduced Stmn2 expression impairs synaptic vesicle trafficking in midbrain neurons. [0016] Genetic and genomic studies have advanced the understanding of the pathogenesis of inherited PD; however, the pathophysiology of idiopathic PD remains unclear due to lack of integrated approach for large-scale multi-dimensional data. Herein, a novel multiscale network approach is described to establish transcriptomic network from postmortem PD brain data. The analysis delineates structures of gene-gene regulatory networks in PD and identifies novel network regulators that are functionally connected to previously identified PD risk genes. STMN2 , identified as encoding a neuron-specific stathmin family protein and down- regulated in PD brains, as the top regulator of the transcriptomic network underlying PD. Perturbation of Stmn2 expression in mice validates its regulatory role. Stmn2 deficient mice show dopaminergic neuron vulnerability, phosphorylated α-synuclein elevation, and locomotor function deficits. As predicted from the network analysis, reduced Stmn2 expression impairs synaptic vesicle trafficking in midbrain neurons. The present disclosure sheds light on the complexity of PD pathogenic network and thus facilitates identification of novel PD therapeutic targets.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0018] FIGS. 1 A-1D show differentially expressed genes (also referred to herein as “DEG” and/or “DEGs”) and genes correlated with Braak score (also referred to herein as “BCG” and/or “BCGs”) in the substantia nigra of PD patients. FIG. 1A shows gene expression of BCGs in GSE49036 with Braak stage indicated on top of the heatmap and DEG status on the side. FIG. IB shows overlap of BCGs and DEGs. FIG. 1C shows top enriched GO terms associated with positively and negatively correlated BCGs, respectively. FIG. ID shows top enriched GO terms associated with downregulated DEGs only. Upregulated DEGs were not significantly associated with any GO terms.

[0019] FIGS. 2A-2G show the results of Multiscale Embedded Gene co-Expression

Network Analysis (“MEGENA”), which identifies modules most associated with PD. In FIG. 2A, the global MEGENA network was rank ordered with highlighted modules associated with PD. In FIG. 2B, the MEGENA modules based on enrichment with DEG/BCG signatures are shown. FIG. 2C shows overall module enrichment with DEGs. FIG. 2D shows overall module enrichment with BCGs. FIG. 2E shows overall module enrichment with cell-type signatures. In FIG. 2F, major pathways associated with modules were annotated. In FIG. 2G, the top-ranked module M88 was enriched with down-regulated DEGs and negative BCGs and was neuron-specific. Key regulators were highlighted with burgundy circles. The pie chart of each node indicates whether it is a DEG, BCG or GW AS, respectively, with blue/light purple for downregulation/negative correlation, red/light coral for upregulation/positive correlation. The size of the node corresponds to the importance/ranking of the nodes.

[0020] FIGS. 3A-3E show that network neighborhoods of the top-ranked PD GWAS hits are enriched for the PD DEG/BCG signatures. In. FIG. 3 A, ranking of PD GWAS hits based on network neighborhood enrichment with DEGs/BCGs is shown. FIGS. 3B-3E show network neighborhoods of BDNF (FIG. 3B), INPP5F (FIG. 3C), AHR (FIG. 3D), and HSD17B1 (FIG. 3E), respectively.

[0021] FIGS. 4A-4C show that RNA sequencing of the midbrain samples from Stmn2-

AAV injected mice validates network structure. FIG. 4 A is a volcano plot that shows genes that are significantly differentially expressed in the midbrain between mice receiving scrambled AAV or Stmn2-shRNA AAV. FIG. 4B shows overlap of STMN2-correlated genes in PD patients and DEG signatures from Stmn2-knockdown mice. FIG. 4C shows that genes within 2-layer neighborhood of STMN2 are significantly enriched with downregulated DEGs identified in Stmn2 knockdown mouse midbrain. Nodes with burgundy labels are key hubs in MEGENA network.

The left upper quarter of each node represents whether it is a DEG in human PD postmortem brains and right upper quarter represents whether it is a DEG in mice with Stmn2 knockdown. The left lower quarter indicates whether it is a PD GWAS gene and the right lower quarter indicates whether it is correlated with STMN2 in PD patients.

[0022] FIGS. 5A-5I are locomotor behavioral analysis and striatal characterization in mice with AAV-shRNA mediated knockdown of Stmn2. FIG. 5A is a schematic showing the experimental plan. The knockdown efficiency of the AAV shRNA-Stmn2 was tested in N2A cells as shown in FIG. 11 A-l 1C. AAV carrying scrambled shRNA or Stmn2-targeting shRNA was injected in the right substantia nigra. Behavioral experiments were performed from Day 22 to Day 27 post injection. FIG. 5B shows that the injected mice were examined in the rotarod test which was composed of 3 trials. The average of 3 trials was used to represent the performance of each individual mouse. N=15 in each group. Student’s t-test, two-sided, t = 2.873, df = 28, p-value = 0.008. FIGS. 5C-5E show that the injected mice were examined by open field experiment. The vertical episode count (FIG. 5C, t = 2.1851, df = 28, p-value = 0.038), total distance (FIG. 5D, t = - 0.36436, df = 28, p-value = 0.3592) and center distance legacy (FIG. 5E, t = 1.7304, df = 28, p- value = 0.094) were compared between the control and Stmn2 knockdown mice. N=15 in each group, two-sided Student’ s t-test for two group comparison. In FIG. 5F, the injected mice were also examined by open field experiment at baseline, under saline injection and under amphetamine injection. The counter-clockwise revolution was compared between the control group and Stmn2 knockdown group. N=15 in each group, two-way ANOVA, df=l, F=75.951, p=2E-16 between control and Stmn2 knockdown; df=14, F=13.669, p= 2e-16 between time points and df=14, F=6.244, p=2.27E-l 1 for interacting terms; post-hoc comparison was performed using Tukey HSD test *<0.05, **<0.01. In FIG. 5G, the content of dopamine (“DA”) and its major metabolites (3- MT, DOPAC and HVA) were measured in the contralateral and ipsilateral striatal tissues isolated from mice that received scrambled AAV and Stmn2-shRNA AAV injections, N=7 in each group. The biogenic monoamine levels of the ipsilateral striatum were normalized to those of the contralateral side in each individual mouse and the comparison between mice with scrambled and Stmn2-shRNA AAV injection was analyzed by two-sided Student’s t-test. DA: t = 4.4367, df = 12, p-value = 0.0008; DOPAC: t = 2.8365, df = 12, p-value = 0.01499; 3-MT: t = 2.614, df = 12, p- value = 0.02263; HVA: t = 3.6415, df = 12, p-value = 0.003379. In FIG. 5H, anti-DAT antibody was used to detect DAT+ terminals in the striatum in mice that received scrambled AAV and Stmn2-shRNA AAV injections. RFP serves as an indicator of transfected terminals. FIG. 51 shows a graph depicting the quantitative analysis of DAT+ immunofluorescent intensity in the two groups of mice. The DAT fluorescence intensity of the ipsilateral striatum was normalized to that of the contralateral side in each individual mouse. N=3 in each group. Two-sided Student’s t-test. t = 2.9255, df = 4, p-value = 0.04.

[0023] FIGS. 6A-6G show pathological characterization in the substantia nigra and striatum of mice with Stmn2 knockdown. FIG. 6A shows immunohistochemistry staining with anti-TH antibody in the substantia nigra from mice injected with AAV carrying scrambled or Stmn2 shRNAs. FIG. 6B shows stereological counting of TH+ neurons in mice injected with AAV carrying scrambled or Stmn2 shRNAs was analyzed by two-sided Student’s t-test. N=3 in each group t = 4.357, df = 4, p-value = 0.012. FIG. 6C depicts immunofluorescent staining with anti-cleaved caspase-3 and TH antibody in the substantia nigra of mice injected with AAV carrying scrambled or Stmn2 shRNA indicated apoptotic neuronal death induced by Stmn2 knockdown. FIG. 6D shows immunofluorescent staining with anti-pS129 of α-synuclein antibody in the substantia nigra of mice injected with AAV carrying scrambled or Stmn2 shRNA. FIG. 6E shows a western blot of the midbrain tissues isolated from mice received either scrambled AAV or Stmn2 knockdown AAV. FIG. 6F shows densitometry quantification of pS129/total alpha synuclein in the control and Stmn2 knockdown group. N=3 in each group, two-sided Student’s t- test. t = -4.2067, df = 4, p-value = 0.0136. FIG. 6G shows densitometry quantification of Stmn2/actin in the control and Stmn2 knockdown group. N=3 in each group, two-sided Student’s t-test. t =3.6018, df = 4, p-value = 0.0227

[0024] FIGS. 7A-7C show pHluorin based analysis of SV endo- and exocytosis in primary midbrain DAergic neuron cultures with Stmn2 knockdown. In FIG. 7A, Stmn2 knockdown in midbrain DAergic neurons slowed SV endocytosis but the exocytic fraction remained unchanged. Two-sided Student’s t-test for two group comparison t = -2.6572, df = 19, p-value = 0.01556 for endocytosis time constant; t=0.87198, df=19, p=0.3941. FIG. 7B shows that stmn2 knockdown in midbrain DAergic neurons barely affected SV exocytosis time constant. This is a subset of neurons recorded in (FIG. 7A). Two-sided Student’s t-test for two group comparison t = -1.6213, df = 9, p-value = 0.1394. In FIG. 7C, stmn2 knockdown in midbrain DAergic neurons also slowed SV exocytosis during stimulation. This is a subset of neurons recorded in FIG. 7A and the same neurons in FIG. 7B. Two-sided Student’s t-test for two group comparison t = 3.6414, df = 9, p-value = 0.005389. N indicated in each experiment. [0025] FIG. 8 depicts the architecture of the systemic network approach for PD. RNA expression profiles of healthy and PD postmortem human brains were collected from GEO database. From the microarray RNA expression data, first differentially expressed gene signatures between the controls and PD patients were identified. Then the control and PD samples were merged into one single dataset, respectively, by Z-score transformation with corrections for covariates such as gender. The merged data were used for co-expression network construction and causal network inference. Modules identified in the co-expression network were overlapped with DEG signatures and cell-type specific markers for prioritization. The causal network topology was then incorporated to identify key driver genes for the top modules. Finally, the top-ranked key driver genes were selected for experimental validation.

[0026] FIG. 9 illustrates that STMN2 is downregulated in the substantia nigra of postmortem PD brain. Standardized mean difference of STMN2 expression in the substantia nigra of PD patients in 8 human studies was shown. A fixed effect model was applied based on heterogeneity test. [0027] FIG. 10 shows immunostaining of Stmn2 in mouse substantia nigra. Stmn2 expression was found abundant in TH+ neurons in the brain slices in 3 -month-old wildtype C57/bl6 mice.

[0028] FIGS. 11 A-l 1C illustrate knockdown efficiency of Stmn2-shRNA construct and

AAV. In FIG. 11 A, the efficiency of two STMN2-shRNA constructs was tested by western blot in N2A cells. Two biological replicates in each experiment and three independent experiments were performed. One-way ANOVA and TukeyHSD test were used to compare the differences between groups. F=43.27, df=2, p=5.9E-07; Stmn2KD A- Scrambled, p=lE-06; Stmn2KDB-Scrambled, p=6E-06. In FIG. 1 IB, immunostaining of STMN2 in the wildtype and STMN2 knockdown midbrain cultures is shown. Construct B was used in all following experiments and the knockdown efficiency of STMN2 was consistent with what was observed in N2A cells. Two- sided Student’s t-test, t= 3.4909, df = 19, p-value = 0.0024. FIG. 11C shows the knockdown efficiency of Stmn2-shRNA AAV at 72 hours post-infection in N2A cells.

[0029] FIG. 12 shows the expression of ENST00000220876 stratified by CDR.

[0030] FIG. 13 shows the expression of ENST00000518111 stratified by CDR.

[0031] FIG. 14 shows the overexpression of Stmn2 in a-syn inducible PC12 cell lines.

Wild type (left panel), A30P (middle panel), and A53T (right panel) a-synuclein were induced by 1 μg/ml Dox in PC 12 cell line after splitting cells into 6-well plates. 24 hours later, CMV-driven Stmn2 overexpression plasmid was transfected into these cells with increasing dose (with GFP plasmid: 1 : 1, 2: 1 and 4:1 ratio, making the total amount of DNA the same among groups). 48 hours after transfection, cells were collected and cell lysates were subjected to western blot analysis.

[0032] FIGS. 15A-15D show open field results for in vivo experiments of Synaspin-

Stmn2-IRES-EGFP into heterozygous Thy-1 α-synuclein mice as compared control mice. FIG. 15A shows open field total distance traveled. FIG. 15B shows open field center distance traveled. FIG. 15C shows open field total rearing frequency. FIG. 15D shows open field center rearing frequency. *N values represent number of animals at first assessment.

DETAILED DESCRIPTION

[0033] A first aspect of the present disclosure relates to a method for treating a neurodegenerative disorder characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites in an individual diagnosed with or suspected of having a loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. The method includes administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2.

[0034] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present invention are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] It is further appreciated that certain features described herein, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub- combination.

[0036] As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0037] As used herein, the term “purified” means that when isolated, the isolate contains at least 90%, at least 95%, at least 98%, or at least 99% of a compound described herein by weight of the isolate.

[0038] The term “cell or group of cells” is intended to encompass single cells as well as multiple cells either in suspension or in monolayers. Whole tissues also constitute a group of cells.

[0039] In accordance with this and all aspects of the present disclosure, a subject suitable for treatment using the methods of the present disclosure includes any animal, preferably a mammal, e.g., human, non-human primate, mice, rats, other rodents, cat, rabbit, dog, cattle, horse, sheep, swine, goat, deer, elk, bison, etc. Preferably, the subject is a human.

[0040] A neurodegenerative disorder as described herein may be characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. A neurodegenerative disorder as described herein may occur in an individual diagnosed with or suspected of having a loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. [0041] Neurodegenerative disorders may cause cognitive impairment. Cognitive impairment as described herein includes any impairment to thought processes, including, for example, loss of higher reasoning, forgetfulness, learning disabilities, concentration difficulties, decreased intelligence, and any other reduction in mental function. Cognitive impairment may be present at birth or can occur at any point in a person’s lifespan. Accordingly, suitable subjects for treatment in accordance with the methods of the present disclosure include infants, children, adolescents, young adults, adults, and elderly. In one embodiment, the subject is of advanced age or is elderly.

[0042] Neurodegenerative disorders may also result in one or more physical manifestations of degeneration including, for example, shaking, stiffness, and difficulty with walking, balance, and coordination, which may be caused by PD.

[0043] The treatment of a neurodegenerative disorder as described in the present aspect may be useful in improving cognition of a subject, for example, in a subject having one or more neurodegenerative disorders. In one embodiment, the neurodegenerative disorder is selected from the group consisting of Parkinson’s Disease, Alzheimer’s Disease, frontotemporal dementia, senile dementias, dementia with lewy bodies, and may result in mild, moderate, or severe cognitive impairment. In one embodiment, the neurodegenerative disorder is Parkinson’s Disease (PD).

[0044] An agent capable of increasing the expression and/or activity of STMN2 as described herein includes, for example, a vector comprising a nucleic acid encoding STMN2. A vector as described herein may include a nucleic acid encoding for STMN2 from a variety of organisms, including, for example, mouse and/or human.

[0045] Mouse Stmn2 gene encodes, for example, one transcript (Accession No.

NM_025285.2 Accession No. NP_079561.1 stathmin-2, ENSMUSP00000029002) and the protein is, in one embodiment, identical to human isoform 2 (Accession No.

NM_007029.3 → Accession No. NP_008960.2, ENSP00000220876) on the amino acid level, which has been cloned into the plasmid described herein. The human STMN2 gene encodes two isoforms: (1) isoform 1 (Accession No. NM_001199214.1 → Accession No.

NP_001186143.1, ENST00000518111) which is a longer transcript but has low expression, and (2) isoform 2. Isoform 2 uses an alternative exon and has a distinct C terminal, which is, in one embodiment, more abundantly expressed. The Synapsin_Stmn2_IRES__GFP may be packaged into AAV9 for overexpression in administration into, for example, the mouse brain, and, may, in one embodiment, have titer around approximately 10¹³ gc/ml. [0046] Examples of mouse Stmn2 sequences include, for example, Accession No.

NM_025285.2, Accession No. NP_079561.1, and ENSMUSP00000029002 (transcript). Mouse

Stmn2 sequence (Accession No. NM_025285.2) is shown below in SEQ ID NO: 1. The shRNA targeting region is bolded.

In one embodiment, the nucleic acid encoding STMN2 has the nucleotide sequence of SEQ ID NO: 1, or Accession No. NM_025285.2.

[0047] Mouse Stmn2 protein sequence (Accession No. NP_079561.1) is shown below in

SEQ ID. NO: 2.

[0048] Examples of human Stmn2 Isoform 1 sequences include, for example, Accession

No. NM_001199214.2, Accession No. NP_001186143.1, and ENST00000518111 (transcript). Homo sapiens Stmn2 Isoform 1 sequence (Accession No. NM_001199214.1) is shown below in SEQ ID NO: 3.

[0049] Homo sapiens Stmn2 Isoform 1 protein sequence (Accession No.

NP_001186143.1) is shown below in SEQ ID. NO: 4.

[0050] Examples of human Stmn2 Isoform 2 sequences include, for example, Accession

No. NM_007029.3, Accession No. NP_008960.2, and ENSP00000220876 (transcript). Homo sapiens Stmn2 Isoform 2 sequence (Accession No. NM_007029.3) is shown below in SEQ ID

NO: 5. The shRNA targeting region is bolded.

[0051] In one embodiment, the nucleic acid encoding STMN2 has the nucleotide sequence of SEQ ID NO: 5, or Accession No. NM_007029.3.

[0052] Homo sapiens Stmn2 Isoform 2 protein sequence (Accession No. NP 008960.2) is shown below in SEQ ID. NO: 6.

[0053] A sequence alignment is provided in Table 1, comparing mouse Stmn2

(Accession No. NM_025285.2), SEQ ID NO: 1, to Homo sapiens Stmn2 Isoform 2 (Accession No. NM_007029.3), SEQ ID NO: 5.

[0054] Accordingly, in one embodiment of the present disclosure, the vector comprises a nucleic acid encoding STMN2 comprising the sequence of any of SEQ ID NOS: 1-6, in particular SEQ ID NO: 1 and/or SEQ ID NO: 5. In another embodiment of the present disclosure, the vector comprises a nucleic acid encoding STMN2 comprises an amino acid sequence having about 70-80% sequence similarity to SEQ ID NO:l and/or SEQ ID NO: 5, more preferably, about 80-90% sequence similarity to SEQ ID NO:l and/or SEQ ID NO: 5, and more preferably 90-95% sequence similarity to SEQ ID NO:l and/or SEQ ID NO: 5, and most preferably about 95-99% sequence similarity to SEQ ID NO:l and/or SEQ ID NO: 5. Thus, unless indicated to the contrary, sequences having less than 100% similarity with SEQ ID NO: 1 and/or SEQ ID NO: 5 may be used in the present disclosure.

[0055] The sequences of the present disclosure may differ from the native polypeptides designated as SEQ ID NOS: 1-6 in terms of one or more additional insertions, substitutions or deletions, e.g., one or more amino acid residues within SEQ ID NO: 1 and/or SEQ ID NO: 5 may be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. That is to say, the change relative to the native sequence would not appreciably diminish the basic properties of the sequence. Substitutions may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0056] In other embodiments, non-conservative alterations (e.g., one or amino acid substitutions, deletions and/or additions) can be made. Molecular alterations may be accomplished by methods known in the art, including primer extension on a plasmid template using single stranded templates (Kunkel et al., “Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection,” Proc. Acad. Sci., USA 82:488-492 (1985), which is hereby incorporated by reference in its entirety), double stranded DNA templates, and by PCR cloning (Braman, J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa,

N. J. (2002), which is hereby incorporated by reference in its entirety).

[0057] Homology as described herein includes the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Homology can alternatively be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA or two polypeptide sequences may be “substantially homologous” to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecules.

[0058] As used herein, “isolated” protein or polypeptide refers to a protein or polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Purity can be measured by any appropriate standard method, for example, by HPLC analysis, column chromatography, or polyacrylamide gel electrophoresis. An isolated protein or polypeptide of the disclosure can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods. As used herein, the phrase “substantially isolated” means a compound that is at least partially or substantially separated from the environment in which it is formed or detected.

[0059] In one embodiment, the expression vector containing a nucleic acid encoding

STMN2 is an adeno-associated viral vector (“AAV”). An AAV vector as described herein includes any vector derived from an adeno-associated virus serotype, including, without limitation, AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and any combination thereof. In one embodiment, the AAV is AAV9. An AAV vector may have one or more of the AAV wild-type genes deleted in whole or in part, for example the Rep and/or Cap genes, but still retain functional flanking inverted terminal repeat (“ITR”) sequences. Functional ITR sequences may be necessary for rescue, replication, and packaging of an AAV virion. Accordingly, an AAV vector may include at least those sequences that work to rescue, replicate, and package (e.g., functional ITRs) of the virus. ITRs may be altered and need not be the wild-type nucleotide sequences (e.g., by the insertion, deletion or substitution of nucleotides) so long as the sequences provide the ability to rescue, replicate, and package. Various adenovirus-based gene delivery systems have been developed and will be understood by those skilled in the art. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are suited for gene transfer because they grow easily, are easily manipulated, and exhibit a broad host range both in vivo and in vitro. Adenovirus may be produced at high titers and may be stable for purification and storage. Adenoviruses generally cause only low level morbidity and are generally not associated with human malignancies. Various adenovirus-based gene delivery systems are described in the art, see, for example, Haj- Ahmad et al., “Development of a Helper-Independent Human Adenovirus Vector and Its Use in the Transfer of the Herpes Simplex Virus Thymidine Kinase Gene,” J. Virol. 57:267-274 (1986); Bett et al., “Packaging Capacity and Stability of Human Adenovirus type 5 vectors,” J. Virol. 67:5911-5921 (1993); Mittereder et al., “Evaluation of the Efficacy and Safety of In Vitro , Adenovirus-Mediated Transfer of the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA,” Human Gene Therapy 5:717-729 (1994); Seth et al., “Mechanism of Enhancement of DNA Expression Consequent to Cointernalization of a Replication-deficient Adenovirus and Unmodified Plasmid DNA,” J. Virol. 68:933-940 (1994); Barr et al., “Efficient Catheter-Mediated Gene Transfer Into the Heart Using Replication-Defective Adenovirus,” Gene Therapy 1:51-58 (1994); Berkner, K. L., “Development of Adenovirus Vectors for the Expression of Heterologous Genes,” BioTechniques 6:616-629 (1988); and Rich et al., “Development and Analysis of Recombinant Adenoviruses for Gene Therapy of Cystic Fibrosis,” Human Gene Therapy 4:461-476 (1993), all of which are hereby incorporated by reference in their entirety. Construction of recombinant adeno-associated virus (also referred to herein as “rAAV”) vectors is known, see, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941; WO 92/01070; WO 93/03769; Lebkowski et al., “Adeno- Associated Virus: A Vector System for Efficient Introduction and Integration of DNA Into a Variety of Mammalian Cell Types,” Molec. Cell. Biol. 8:3988-3996 (1988); Carter, B. J., “Adeno-associated virus vectors,” Current Opinion in Biotechnology 3:533-539 (1992); Muzyczka, N., “Use of Adeno-Associated Virus as a General Transduction Vector for Mammalian Cells,” Current Topics in Microbiol and Immunol. 158:97-129 (1992); and Kotin, R. M., “Prospects For the Use of Adeno-Associated Virus as a Vector for Human Gene Therapy,” Human Gene Therapy 5:793-801 (1994), all of which are hereby incorporated by reference in their entirety.

[0060] A recombinant virus includes a virus that has been genetically altered (e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle).

[0061] An AAV virion as described herein includes a complete virus particle, including a wild-type AAV virus particle (i.e., including a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat). Single-stranded AAV nucleic acid molecules of either complementary sense (i.e., “sense” or “antisense” strands) can be packaged into any one AAV virion. [0062] As described herein, transfection may include uptake of foreign DNA by a cell. A cell may be transfected when exogenous DNA has been introduced inside a cell membrane. Various transfection techniques are known in the art, see, for example, Graham et al. “A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA,” Virology 52:456 (1973) and Chu et al., “SV40 DNA Transfection of Cells in Suspension: Analysis of Efficiency of Transcription and Translation of T-Antigen,” Gene 13:197 (1981), both of which are hereby incorporated by reference in their entirety. Techniques can be used to introduce one or more exogenous DNA moieties, for example, a plasmid vector and other nucleic acid molecules, into suitable host cells.

[0063] As used herein, the term “heterologous” includes sequences that are not normally joined together and/or are not normally associated with a particular cell. Accordingly, a heterologous region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heterologous as described herein.

[0064] The term “promoter” is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, where the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.

[0065] A therapeutically effective amount as described herein includes therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent, slow down, alleviate, and/or lessen the disease (e.g., neurodegenerative disorder) or its complications, preventing or inhibiting it from manifesting, preventing, or inhibiting it from recurring, merely preventing or inhibiting it from worsening, curing the disease, reversing the progression of the disease, prolonging a patient's life or life expectancy, ameliorating the disease, or a therapeutic effort to effect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful.

[0066] In various embodiments, the agents according to the present disclosure may be formulated for delivery via any route of administration. In one embodiment, the agent is administered to the central nervous system of the individual. The route of administration may refer to any administration pathway known in the art, including but not limited to intraneuronal, intracardiac, aerosol, nasal, oral, transmucosal, transdermal, subcutaneous, or parenteral. Parenteral refers to a route of administration that is generally associated with injection, including intranasal, intrathecal, intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or in the form of lyophilized powders. In one embodiment, the agent is administered intrathecally, intranasally, intraperitoneally, orally, parenterally, nasally, subcutaneously, intravenously, intramuscularly, intracerebroventricularly, intraparenchymally, by intranasal inhalation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. In one embodiment, the agent is administered intrathecally or intranasally.

[0067] A second aspect relates to a method for the treatment of an individual with PD.

The method includes administering to the individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2.

[0068] This aspect is carried out in accordance with the previously described aspect.

[0069] The treatment described herein may, in one embodiment, be administered as part of a pharmaceutical composition and may further include a pharmaceutically acceptable carrier. [0070] Modes of administration of the agents described herein include, in one embodiment, a composition that may be administered as a formulation in combination with one or more pharmaceutically acceptable carrier, excipient, or additive. A carrier, excipient, or additive may be “acceptable” in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient thereof.

[0071] Agents described herein may include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

[0072] In one embodiment, the treatment, when in the form of a composition, further comprises a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carriers” as described herein, refer to conventional pharmaceutically acceptable carriers. See Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), which is hereby incorporated by reference in its entirety (describing compositions suitable for pharmaceutical delivery of the inventive compositions described herein). In particular, a pharmaceutically acceptable carrier as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. In one embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of a liquid filler, a solid filler, a diluent, an excipient, a solvent, and an encapsulating material.

[0073] Pharmaceutically acceptable carriers (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives and solubilizing agents) are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful in the present disclosure include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; adjuvants (selected so as to avoid adjuvant-induced toxicity, such as a (3- glucan as described in U.S. Pat. No. 6,355,625, which is hereby incorporated by reference in its entirety), or a granulocyte colony stimulating factor (GCSF)); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0074] In one embodiment, the composition may further comprise an adjuvant. Suitable adjuvants are known in the art and include, without limitation, flagellin, Freund’s complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.

[0075] A method in accordance with the present disclosure may include bringing into association a composition as disclosed (“active ingredient”) with a carrier which constitutes one or more accessory ingredients. In general, formulations may be prepared by uniformly and intimately bringing into association an active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of an active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

[0076] In some examples, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer’s patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0077] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of an active ingredient therein.

[0078] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render a formulation isotonic with the blood of an intended recipient. Formulations for parenteral administration also may include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0079] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0080] A formulation may include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. A composition, as a formulation, may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990., which is hereby incorporated by reference in its entirety).

[0081] A carrier, excipient, or additive may include a composition to aid in cellular uptake, transportation or transfer of the agent capable of increasing the expression and/or activity of STMN2 across a cell membrane to permit intracellular translation of a protein product therefrom. Various cell penetrating peptides, nanoparticles, lipoplexed configurations, and other carriers for enhanced cellular uptake may be used. Commercially available examples include RNA IMAX™, MESSENGER MAX™, JET MESSENGER™, and TRANS IT™.

[0082] As used herein, the term “reference level” refers to an amount of a substance, e.g., particular cell type (for example, neurons), which may be of interest for comparative purposes.

In some embodiments, a reference level may be the level or concentration of a population of a cell type expressed as an average of the level or concentration from samples of a control population of healthy (disease-free and/or pathogen-free) subjects. In other embodiments, the reference level may be the level in the same subject at a different time, e.g., before the present disclosure is employed, such as the level determined prior to the subject developing a disease (e.g., a neurodegenerative disorder), disease condition, and/or pathogenic infection, prior to initiating therapy, such as, for example, the methods described herein, or earlier in the therapy. Mammalian subjects according to this aspect of the present disclosure include, for example, human subjects, equine subjects, porcine subjects, feline subjects, and canine subjects. Human subjects are preferred.

[0083] For purposes of this and other aspects of the disclosure, the target “subject” encompasses any vertebrate, such as an animal, preferably a mammal, more preferably a human. In the context of administering a composition of the disclosure for purposes of increasing the expression and/or activity of STMN2, a target subject encompasses any subject that has or is at risk of having a neurodegenerative disorder (e.g., PD) (as compared to a reference level), optionally characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites. Particularly susceptible subjects include adults and elderly adults. However, any infant, juvenile, adult, or elderly adult that has or is at risk of having any of the conditions described herein can be treated in accordance with the methods of the present disclosure. In one embodiment, the subject is an infant, a juvenile, or an adult.

[0084] As used herein, the phrase “therapeutically effective amount” means an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. The therapeutic effect is dependent upon the disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject’s response to treatment. The term “treatment” or “treat” may include effective inhibition, suppression, or cessation of a neurodegenerative disorder characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites, so as to prevent or delay the onset, retard the progression, or ameliorate the symptoms of the neurodegenerative disorder (as compared to a reference level). [0085] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least one additional agent beyond the agents described herein, optionally, by the same route and at the same time or at substantially the same time. As used herein, the term “separate” therapeutic use refers to an administration of at least one additional agent beyond the agents described herein at the same time or at substantially the same time by different routes. As used herein, the term “sequential” therapeutic use refers to administration of at least one additional agent beyond the agents described herein at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of the additional agent before administration of the agents described herein. It is thus possible to administer the additional agent over several minutes, hours, or days before applying the agents described herein.

[0086] A third aspect of the present disclosure relates to a vector comprising a nucleic acid encoding STMN2.

[0087] This aspect is carried out in accordance with the previously described aspects.

[0088] In one embodiment, the vector is an adeno-associated viral vector (AAV). In one embodiment, the AAV is AAV9. The vector is carried out in accordance with the previously described aspects.

[0089] In one embodiment, the STMN2 is human. In another embodiment, the STMN2 is murine. In one embodiment, the human STMN2 comprises the nucleotide sequence of SEQ ID NO: 5, as described herein. In another embodiment, the murine STMN2 comprises the nucleotide sequence of SEQ ID NO: 1, as described herein.

[0090] In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the disclosure, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present disclosure. The following description of example embodiments is, therefore, not to be taken in a limited sense.

[0091] The present disclosure may be further illustrated by reference to the following examples. EXAMPLES

[0092] The following examples are intended to illustrate, but by no means are intended to limit, the scope of the present disclosure as set forth in the appended claims.

Example 1 - Materials and Methods.

[0093] Cells and Animals - All animal procedures were performed under protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Care and Use Committee. [0094] Mouse N2A neuroblastoma cells were cultured in DMEM supplemented with

10% FBS at 37°C with 5% CO2. Approximately 80,000 cells were seeded into 12 well plates 24 hours before lipofectamine 3000 (Life Technology) transfection. 72 h after transfection, cells were collected in RIP A buffer for Western Blot.

[0095] C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Maine) and housed in the pathogen-free Center for Comparative Medicine at Mount Sinai School of Medicine. Handling procedures were in accordance with National Institutes of Health guidelines and approved by the Mount Sinai Institutional Animal Care and Use Committee (IACUC). In addition to the breeding pairs for midbrain primary cultures, thirty 2-month-old male C57BL/6J mice were randomized into two groups that received AAV injection of either scrambled or Stmn2 shRNA for in vivo Stmn2 knockdown experiments and behavior assays.

[0096] Mouse midbrain neurons were cultured and stimulated as previously described

(Mani et al., “Live Imaging of Synaptic Vesicle Release and Retrieval in Dopaminergic Neurons,” Frontiers in Neural Circuits 3:3 (2009) and Pan et al., “Calbindin Controls Release Probability in Ventral Tegmental Area Dopamine Neurons,” Nat. Neurosci. 15:813-815 (2012), both of which are hereby incorporated by reference in their entirety). Typically, four P0-P1 mouse brains are required for a MB culture. Calcium phosphate was used for transfection to achieve sparse expression and to ensure analysis of single neurons during the imaging experiments. Transfection was carried out at DIV 3-5, after which, the growth medium was replaced with a fresh medium supplemented with an antimitotic agent, ARA-C (Sigma-Aldrich) and Glial cell line-Derived Neurotrophic Factor (GDNF) (Millipore).

[0097] Data collection and pre-processing Publicly available datasets comparing the expressional difference between Parkinson’s disease patients and controls were downloaded from Gene Expression Omnibus (Wheeler et al., “Database Resources of the National Center for Biotechnology Information,” Nucleic Acids Research 29:11-16 (2001), which is hereby incorporated by reference in its entirety). The gene expression profile of each dataset was quantile normalized and corrected for gender and brain region etc., if the information was available. Datasets with sample size greater than 6 in each of PD and normal control group were selected for differential gene expression analysis and datasets with sample size greater than 15 in each group were selected for network construction.

[0098] Differential gene expression analysis Student’s t-test was applied to each gene between patients and controls. Permutation-based false discovery rate (FDR) was used to control multiple testing errors. Genes with fold change greater than 1.2 (or less than 0.83) and FDR < 0.05 were considered differentially expressed genes (DEG). DEGs from individual dataset were combined to generate the overall DEG signature by a directional voting method, where DEGs that occurred in at least 30% of the data sets were included.

[0099] Network construction Each pre-processed dataset was split into disease and control groups. Z-score transformation was applied to remove batch effect and to allow data merging. The merged cohort contained expression data from 165 PD samples and 131 controls. Module detection was done using the Weighted Gene Co-expression Network Analysis (WGCNA) (Bagot et al., “Circuit-Wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility,” Neuron 90:969-983 (2016); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); Zhang et al., “A General Framework For Weighted Gene Co-Expression Network Analysis,” Statistical Applications in Genetics and Molecular Biology 4:Articlel7 (2005); and Zhang et al., “Characterization of Genetic Networks Associated with Alzheimer’s Disease,” Methods in Molecular Biology 1303:459-477 (2016), all of which are hereby incorporated by reference in their entirety). Bayesian networks (Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013), which is hereby incorporated by reference in its entirety) were constructed based on the merged expression profiles. The links presenting in 30% of all networks were considered true links in the final Bayesian network. Key driver analysis (“KDA”) (Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013), which is hereby incorporated by reference in its entirety) was performed to identify the central causal genes regulating a large number of other genes.

[00100] Gene set enrichment analysis Modules were overlapped with Gene Ontology (“GO”) terms, signaling pathways, and cell type specific expression gene signatures (FIGS. 1 A- ID). The significance level was determined by hypergeometric test and Benjamini-Hochberg (BH) correction for multiple testing.

[00101] Western blot Sample concentration was determined by BCA (Pierce, Waltham, MA). Then samples were boiled in 3X sample buffer at 95°C for 10 min and briefly centrifuged. 30ug protein samples were loaded into 4-12% gradient gels (Invitrogen, Carlsbad, CA) and run at 120V in IX MOPS running buffer. Transfer was done at 4°C in transfer buffer with 10% methanol for 2 h at 90V. The membrane was blocked in LICOR blocking buffer for 1 h and then overnight incubation with primary antibody at 4°C. Then the membrane was washed three times in TBST buffer and incubated in fluorescence-conjugated secondary antibody (1:10000, LI- COR, Germany) for 1 h. The membrane was washed three times in TBST buffer with 0.01% SDS followed by imaging in LI-COR imaging system.

[00102] Phluor in-based optical assay for endocytic and exocytic kinetics The phluorin- based optical assay was adopted from (Onoa et al., “Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways,” The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 30:7917-7927 (2010) and Sankaranarayanan et al., “Calcium Accelerates Endocytosis of vSNAREs at Hippocampal Synapses,” Nature Neuroscience 4:129-136 (2001), both of which are hereby incorporated by reference in their entirety) and performed on DIV12-16 for midbrain cultures. For live cell imaging, cells were mounted on a custom-made laminar-flow stimulation chamber with constant perfusion (at a rate of -0.2-0.3 mL / min) of a Tyrode’s salt solution containing (in mM) 119 NaCl, 2.5 KC1, 2 CaC12, 2 MgC12, 25 HEPES, 30 Glucose, 10 mM 6-cyano-7- nitroquinoxaline- 2,3-dione (CNQX), and 50 μM D, L-2-amino-5-phosphonovaleric acid (AP5) and buffered to pH 7.40. NH4C1 solution containing (in mM) 50 NH4C1, 70 NaCl, 2.5 KC1, 2 CaC12, 2 MgC12, 25 HEPES, 30 Glucose, 10 μM CNQX, and 50 μM AP5 and buffered to pH 7.40 was used to reveal total pHluorin expression for normalizing exocytosis. All chemicals were purchased from Sigma-Aldrich except for bafilomycin A1 (1 μM, Calbiochem, 196000-1 SET). Temperature was clamped at 30.0 °C at the objective throughout the experiment. Field stimulations were delivered at 10 V / cm by A310 Accupulser and A385 stimulus isolator (World Precision Instruments). 1 ms pulse was used to evoke single action potentials. Images were acquired using a highly sensitive, back-illuminated EM-CCD camera (iXon+ Model # DU-897E-BV, Andor Corp., CT, USA). Olympus 1X73 microscope was modified for laser illumination. A solid-state 488 nm OPSL smart laser at 50 mW (used at 10% and output at - 2 mW at the back aperture) was built into a laser combiner system for millisecond on/off switching and camera blanking control (Andor Corp). pHluorin fluorescence excitation and collection were through an Olympus PLAPON 60X0 1.42 NA objective using 525/50m emission filter and 495LP dichroic filters (Chroma, 49002). Images were sampled at 2 Hz with an Andor Imaging Workstation driven by Andor iQ-CORE-FST (ver 2.x) iQ3.0 software. A train of 100 action potentials was given to the perfused coverslip for measuring endocytosis time constant and deltaF of exocytosis. 50mM NH4C1 (pH 7.4) was used to measure total vesicle pool. Bafilomycin (ImM) was used to block vesicle re-acidification and a train of 1200 AP was given for measurement of recycling pool and exocytosis time constant.

[00103] In vivo Stmn2 knockdown mouse model and behavioral test Thirty 2-month-old male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Maine). The IACUC guidelines were strictly followed during animal maintenance and procedure. Adeno- associated viruses carrying either scrambled shRNA or Stmn2 -targeting shRNA were packaged by the Boston Children’s Hospital Viral Core. 2 ul of virus with a titer of 10¹³ gc/mL were injected into the right substantia nigra. Behavioral tests were performed during the fourth week after injection.

[00104] Rotarod The locomotor function was tested using a rotarod setting with incremental speed from 4 to 40 RPM. The duration of time an individual mouse remained on the rotarod served as the readout of the experiment. The average duration of 3 trials was used to represent the performance of the mice.

[00105] Open field The overall behavioral features were examined by open field test.

Individual mice were placed into the 16” x 16” animal cage of a Versamax monitor system (Accuscan) in a quiet dark room and allowed to move freely for an hour. The mouse horizontal and vertical movement was monitored and recorded by a grid of 32 infrared beams at ground level and 16 elevated (3”) beams. Then saline was administrated by intraperitoneal (IP) injection and the mice were placed back into the same cage for 30 min. Finally amphetamine was administrated by IP injection at a dose of 2.5mg/kg and the mice were monitored for another hour in the cage. A series of parameters including total distance, vertical movement and rotational behavior were analyzed.

[00106] Immunocytochemistry and immunohistochemistry Cells were fixed by 4% PFA for lOmin and permeabilized in PBS with 0.2% TritonX-100 for lOmin. Then cells were blocked in PBS with 5% BSA for 1 hour and incubated with primary antibody diluted in the blocking buffer at 37°C for 1 h. Cells were then incubated with secondary antibody diluted in the blocking buffer at room temperature for 1 h. The cells were washed 4 times with PBS between each step and were incubated in PBS overnight at 4°C before mounting to reduce background. The immunohistochemistry (DAB) was performed according to the manual provided by VECTOR LABORATORIES (Burlingame, CA).

[00107] Quantification and statistical analysis The data analysis was performed by Bioconductor R 3.2.3. Data were represented by mean ±SEM. Student’s t-test was used for between group comparisons. P<0.05 was considered as statistically significant. Detailed analysis, including the N number, statistic test used and p value, was indicated in each figure and figure legend.

Example 2 - A Systemic Multiscale Network Approach to Deconvolute Complexity of PD. [00108] The present disclosure describes the first comprehensive network analysis of human PD. Utilizing gene expression data from 12 separate human PD studies, the inventors created and interrogated a global PD expression set compared to control. They identified over 1500 differentially expressed genes compared to control and analyzed the top 100. Downregulated genes were associated with synaptic transmission and regulator of neurotransmitter level. Bayesian network analysis was used to infer causality to identify key regulators of the PD network. In addition to genes known to play a role in PD, stathmin-2 (STMN2) was identified as the top ranked key driver gene. STMN2 was down regulated in PD cases.

[00109] The present disclosure presents a network biology approach to study PD by assembling multiple datasets in PD into a single unified cohort with sufficient samples for gene network analysis. The integrative network analysis of this PD cohort identified 82 gene modules in PD, revealed causal relationships among genes and predicted STMN2 as a top key driver in a neuron-specific and synaptic transmission related subnetwork most strongly associated with PD. The experimental validation demonstrates the essential function for STMN2 in regulating presynaptic transmission and vulnerability of DA neurons in animal models. Thus the disclosure not only sheds light on the global landscape of gene interaction and regulatory circuits underlying PD pathogenesis, but also begins to provide insight into the key causal molecules that mediate a potential common mechanism for idiopathic PD.

[00110] The limited number of published PD human brain gene expression profiles and relatively small sample size in each study prevented a thorough understanding of gene expression patterns and molecular mechanisms in PD. Here, gene expression profiling data from 12 human PD studies were collected (Table 2). [00111] For this study, an integrated approach was developed, including data processing, network construction and experimental validation, as shown in FIG. 8. For the gene expression data quality control, normalization and correction were performed for known covariates using a linear regression model (Table 2). Differentially expressed genes (DEGs) were identified between case and control by student’s t-test and controlled multiple testing by Benjamini-Hochberg (BH) correction for individual dataset. A directional voting method was used to generate a consensus DEG signature in PD from the DEG signatures identified in each dataset, followed by gene ontology (GO) analysis, which then identified dysregulated pathways in PD. The expression data from all the PD samples in all the studies were merged into a global PD expression dataset (165) by Z-score transformation and the same process was applied to the control samples (131), leading to a global control expression dataset. Subsequently, weighted gene co-expression network analysis (“WGCNA”) to the global PD and control datasets was conducted. WGCNA starts with a Pearson correlation matrix of all gene pairs which was transformed first into an adjacency matrix by a power function and further into a topological matrix (Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013), which is hereby incorporated by reference in its entirety). A dynamic tree cutoff algorithm was employed to identify gene co-expression modules (Langfelder and Horvath, “Defining Clusters From a Hierarchical Cluster Tree: The Dynamic Tree Cut package for R,” Bioinformatics 24:719-20 (2008) and Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013), both of which are hereby incorporated by reference in their entirety). Modules were then ranked by the enrichment for the consensus DEG signature in PD (Wang et al., “Systems Analysis of Eleven Rodent Disease Models Reveals an Inflammatome Signature and Key Drivers,” Molecular Systems Biology 8:594 (2012) and Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013), both of which are hereby incorporated by reference in their entirety). In parallel,

Bayesian causal networks were constructed from the global PD and control datasets for dissecting the PD related modules to identify key drivers for experimental validation. All p values reported herein were corrected for multiple testing unless otherwise specified.

[00112] Over the past two decades, the majority of PD research has focused on individual genes or pathways based on genetic factors but lacked a systematic method capable of identifying relevant molecules or pathways underlying idiopathic PD pathogenesis. Furthermore, Genome- Wide Association Study (“GW AS”) approaches have revealed many risk loci for PD; however, they are limited in informing the disease etiology, and functional characterization of these loci lags behind. The emergence of systems/network biology based upon large genetic and genomic data for other complex diseases such as cancer, Alzheimer’s disease, obesity and diabetes has led to the discovery of novel mechanisms and disease target genes in parallel and in complement with traditional genetic analysis. These studies have revolutionized the research for complex diseases involving multiple layers of factors, including genetics, epigenetics, and environment. Bailey et al., “Genomic Analyses Identify Molecular Subtypes of Pancreatic Cancer,” Nature 531:47-52 (2016); Chen et al., “Variations in DNA Elucidate Molecular Networks That Cause Disease,” Nature 452:429-435 (2008); Emilsson et al., “Genetics of Gene Expression and Its Effect on Disease,” Nature 452:423-428 (2008); Gadaleta et al., “Integration of Gene Expression and Methylation to Unravel Biological Networks in Glioblastoma Patients,” Genet. Epidemiol. 41:136-144 (2017); Vishnubalaji et al., “Genome-Wide mRNA and miRNA Expression Profiling Reveal Multiple Regulatory Networks in Colorectal Cancer,” Cell Death Dis. 6:el614 (2015); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late- Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); Zhang et al., “A Network Medicine Approach to Build a Comprehensive Atlas for the Prognosis of Human Cancer,” Brief Bioinform. 17: 1044-1059 (2016); Zhu et al., “Increasing the Power to Detect Causal Associations by Combining Genotypic and Expression Data in Segregating Populations,” PLoS Comput. Biol. 3:e69 (2007); and Zhu et al., “Integrating Large-Scale Functional Genomic Data to Dissect the Complexity of Yeast Regulatory Networks,” Nature Genetics 40:854-861 (2008), all of which are hereby incorporated by reference in their entirety. The lack of large-scale molecular data from PD post-mortem brains has impeded the application of such a systems approach in PD research. In addition, although many gene expression studies of PD have been reported, the scale of these studies is small and each alone failed to provide sufficient power to derive a holistic picture of PD related pathways or key regulators.

[00113] It was sought to address above unmet challenge in PD research by assembling gene expression profiles of postmortem human brains from 8 publicly available PD studies into one gene expression dataset, which allowed the multiscale gene coexpression network analysis to systematically uncover intrinsic network structures and key regulators of PD. By investigating the neighborhood context of each gene within the network (see, e.g, FIGS. 3 AGE), it was possible to annotate and predict the biological function for particular genes in a disease specific manner, which complements the current GWAS studies and shed light on experimental validation for genes of interest. Network modules most enriched for the molecular changes in PD are involved in synaptic transmission and oxidative phosphorylation, mitochondrion, myelination and response to unfolded protein. Analysis of these modules and the networks in PD further identified a number of key hub genes including STMN2, which has an overall reduced expression in PD brains.

[00114] The significant role for the key hub genes as regulators for PD pathogenic pathways remains to be further clarified in the future. Distinct from many genetic variants that are causal to the disease in human, the hub genes, identified in this PD network, have not been reported to be associated genetically to the disease. However, they are predicted to govern the expressions of many genes in the specific disease networks and therefore are considered “drivers” that can modify disease onset and progression. Thus, while many disease causal genes are considered poor drug targets, the significance of the hub genes is that they offer an opportunity to become novel drug targets. Indeed, key hub gene STMN2 has never previously been implicated in PD pathogenesis. Interestingly, it was found STMN2 closely linked to four GWAS gene hits (BDNF, INPP5F, STK39 and HTR2A) in the same network, suggesting that they are functionally related.

Example 3 - Differentially expressed genes between PD patients and controls are enriched for disease-associated pathways.

[00115] 1538 consensus DEGs were identified between PD cases and controls in the datasets collected. The differential regulation of the top 100 DEGs with highest frequencies across datasets (as shown in FIG. 2 A, left panel) and their expression in 6 major cell types in human brain (Darmanis et al., “A Survey of Human Brain Transcriptome Diversity at the Single Cell Level,” Proceedings of the National Academy of Sciences of the United States of America 112:7285- 7290 (2015) and Zhang et al., “Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse,” Neuron 89:37-53 (2016), both of which are hereby incorporated by reference in their entirety) (FIG. 2A, right panel) were plotted. Also observed was a distinct regulation pattern in different brain regions. Interestingly, the genes downregulated in most datasets were highly expressed in neurons. Also performed was Gene Ontology (GO) analysis and showed that the downregulated genes were enriched for the GO terms such as synaptic transmission (FET p=1.88E-07, 2.21 fold) and regulation of neurotransmitter levels (FET p=8.97E-08, 3.69 fold) while the upregulated genes were enriched for cell cycle regulation (FET p=3.66E-10, 2.22 fold). The result may suggest that neuronal activity particular neurotransmission was impaired in PD and neurogenesis might be upregulated to compensate for neuronal loss (FIG. 2B).

Example 4 - Co-expressed gene modules in PD represent cell-type-specific gene expression and related functions.

[00116] A total 82 PD related modules were identified by WGCNA as previously described (Wang et al., “Systems Analysis of Eleven Rodent Disease Models Reveals an Inflammatome Signature and Key Drivers,” Molecular Systems Biology 8:594 (2012); Zhang et al., “A General Framework For Weighted Gene Co-Expression Network Analysis,” Statistical Applications in Genetics and Molecular Biology 4:Articlel7 (2005); Zhang et al., “Characterization of Genetic Networks Associated with Alzheimer’s Disease,” Methods in Molecular Biology 1303:459-477 (2016); Zhang et al., “Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse,” Neuron 89:37-53 (2016); Zhang et al., “A Network Medicine Approach to Build a Comprehensive Atlas for the Prognosis of Human Cancer,” Brief Bioinform. 17: 1044-1059 (2016), all of which are hereby incorporated by reference in their entirety) (see FIGS. 2A-2G). To quantify the relevance of these modules to PD, the modules were rank-ordered by the enrichment for the above identified DEGs between PD and control (FIG. 3 A). This measurement reflects the degree of molecular dysregulation with respect to PD. The turquoise module was ranked the top due to the most significant enrichment for the PD gene signature (FET p=2.82E-202, 4.83 fold). Also the turquoise module harbors 7 PD GWAS hits (SCNA, VPS35, NSF, RIT2, LAMP2, HIP1R and SLC03A1) (Nalls et al., “Imputation of Sequence Variants for Identification of Genetic Risks for Parkinson’s Disease: A Meta- Analysis of Genome-Wide Association Studies,” Lancet 377:641-649 (2011); Pankratz et al., “Meta- Analysis of Parkinson’s Disease: Identification of a Novel Locus, RIT2,” Ann. Neurol. 71:370- 384 (2012); Pihlstrom et al., “Supportive Evidence For 11 Loci From Genome-Wide Association Studies in Parkinson’s Disease,” Neurobiology of Aging 34:1708.e7-13 (2013); and Liu et al., “Genome-Wide Association Study Identifies Candidate Genes for Parkinson’s Disease in an Ashkenazi Jewish Population,” BMC medical genetics 12: 104 (2011), all of which are hereby incorporated by reference in their entirety) and five of them (except HIP1R and LAMP 2) were differentially expressed between PD and control. The 2^nd ranked blue module (FET p=1.08E-17, 2.25 fold) was involved in receptor activity and was strongly correlated with many other modules (FIG. 3 A). The other top ranked modules were associated with nerve ensheathment (red), poIII transcriptional regulation (black), nucleosome (green-yellow), double-stranded RNA binding (purple), RNA regulation (yellow) and response to protein folding (light cyan).

[00117] The central nervous system (CNS) is composed of multiple types of cells with distinct functions, which contribute differently to PD pathogenesis and progression. It was sought to investigate the cell type specificity of the modules using the gene signatures for 6 major brain cell types including neurons, astrocytes, microglia, endothelial cells, oligodendrocyte precursor cells (OPC) and oligodendrocytes generated from human brain (Darmanis et al., “A Survey of Human Brain Transcriptome Diversity at the Single Cell Level,” Proceedings of the National Academy of Sciences of the United States of America 112:7285-7290 (2015) and Zhang et al., “Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse,” Neuron 89:37-53 (2016), both of which are hereby incorporated by reference in their entirety). In examining the enrichment of the brain cell type specific signatures in the top 17 modules (FIG. 3B), it was found that the turquoise module was enriched for the neuron-specific gene signature (FET p=7.13E-80, 5.55 fold). The brown module was highly enriched for the microglia-specific markers (FET p=3.12E- 125, 13.65 fold) as well as the endothelial cell specific genes (FET p=2.06E-08, 3.08 fold). The green module was enriched for the astrocyte-specific signature (FET p=8.55E-77, 18.52 fold). The GO analysis revealed functional association between modules and specific cell types. For example, the microglia and endothelial cell-enriched brown module was highly associated with inflammatory response (FET p=3.40E-35, 5.13 fold) and immune cell activation (FET p=9.20E- 15, 4.21 fold) while the neuron-enriched turquoise module was enriched for the genes involved in synaptic transmission (FET p=4.35E-14, 2.43 fold). These data suggested that the co- expression network analysis described herein was capable of detecting cell-type-specific gene expression patterns and associated biological functions.

[00118] Network “rewiring” often takes place during disease pathogenesis and progression. By comparing the co-expression networks in PD and control, it was found that although modules could be conserved between two networks, the interconnection strength was differentially regulated between PD and control, which can be characterized by Modular Differential Connectivity (MDC) analysis. Bagot et al., “Circuit-Wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility,” Neuron 90:969- 983 (2016); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); and Zhang et al., “Characterization of Genetic Networks Associated with Alzheimer’s Disease,” Methods in Molecular Biology 1303:459-477 (2016), all of which are hereby incorporated by reference in their entirety. Among the 17 modules with member size greater than 35 in PD, 7 modules showed more than 30% increase of connectivity over the control network while 3 showed more than 30% decrease in connectivity. Fig. 3B showed a subset of modules with gain or loss of connectivity between PD and control. The green-yellow module with a 72% increase of connectivity in PD is associated with nucleosome and chromatin assembly (FET p=7.06E-8, 19.06 fold), indicating irregular transcriptional regulation in the disease state. The light cyan module with a 200% increase of connectivity in PD associated with protein folding (FET p=2.83E-19, 24.81 fold) and other stress such as oxidative stress, suggesting an elevated cellular stress in PD; the blue module with a 34% increase of connectivity in PD is associated with mitochondrial functions (FET p=4.29E-l 1, 2.28 fold), which implicates a mitochondrial dysfunction in PD. On the other hand, the salmon module with a 32% decrease of connectivity in PD involves ribosomal activity (FET p= 4.00E-90, 131.03 fold) and protein biosynthesis (FET p=1.28E-47, 13.67 fold) while the red and magenta modules with 59% and 58% decrease of connectivity in PD, are associated with nerve ensheathment (FET p=3.19E-07, 33.48 fold) and cell maturation (FET p=1.74E-05, 15.48 fold), respectively, suggesting an impaired myelination in PD pathogenesis and progression. The top ranked turquoise module, with a 54% decrease of connectivity in PD, was involved in active transporter activity (FET p=2.86E-21, 4.11 fold), oxidative phosphorylation (FET p=5.05E-17,

5.13 fold) and synaptic transmission (FET p=4.35E-14, 2.43 fold), indicating reduced capacity of signaling transduction as well as disrupted maintenance of ox-redox homeostasis in PD brains. Impairment of these functions and pathways are believed to dominate in neurodegenerative disease pathways (Chinta and Andersen, “Redox Imbalance in Parkinson’s Disease,” Biochim. Biophys. Acta. 1780:1362-67 (2008); Gao and Goldman-Rakic, “Dopamine Modulation of Perisomatic and Peridendritic Inhibition in Prefrontal Cortex,” J Neurosci. 23: 1622-30 (2003); and Guzman et al., “Oxidant Stress Evoked by Pacemaking in Dopaminergic Neurons is Attenuated by DJ-1,” Nature 468:696-700 (2010), all of which are hereby incorporated by reference in their entirety).

Example 5 - Bayesian subnetworks and key drivers of the top-ranked gene modules underlying PD.

[00119] Bayesian networks (BNs) are widely used to infer causality in gene regulatory networks. Wang et al., “Systems Analysis of Eleven Rodent Disease Models Reveals an Inflammatome Signature and Key Drivers,” Molecular Systems Biology 8:594 (2012); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); and Zhu et al., “Integrating Large-Scale Functional Genomic Data to Dissect the Complexity of Yeast Regulatory Networks,” Nature Genetics 40:854-861 (2008), all of which are hereby incorporated by reference in their entirety. Towards this end, BNs were constructed for the global PD and control datasets and further used the BNs to dissect the gene co-expression modules to derive module specific Bayesian subnetworks that were further used to identify key driver genes based on centrality measurement. Wang et al., “Systems Analysis of Eleven Rodent Disease Models Reveals an Inflammatome Signature and Key Drivers,” Molecular Systems Biology 8:594 (2012); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); and Zhu et al., “Integrating Large-Scale Functional Genomic Data to Dissect the Complexity of Yeast Regulatory Networks,” Nature Genetics 40:854-861 (2008), all of which are hereby incorporated by reference in their entirety. It was found the Bayesian subnetworks of 4 top ranked modules including turquoise (FIG. 4A), blue (FIG. 4B), red (FIG. 4C) and green-yellow. The Bayesian subnetwork of the turquoise module, which is enriched for neuron-specific marker genes (FET p=7.13E-80, 5.55 fold), harbors well-known genetic causes of familial PD such as ATP13A2 ( PARK9 ) and UCHL1 ( PARK5 ) as driver genes (FIG. 4A). Notably, two of the key drivers in the 2^nd ranked blue module, UQCRC2 and ATP5F1, encode mitochondrial proteins in the respiratory chain complex III and V, respectively (FIG.4B). Although UQCRC2 was not differentially expressed between PD patients and controls, deficiency in Cl and CII has been linked to idiopathic PD (Grunewald et al., “Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons,” Annals of Neurology 79:366-378 (2016), which is hereby incorporated by reference in its entirety). The other key driver, SRP54 , was reported to regulate Tau exon 10 alternative splicing (Wu et al., “SRp54 (SFRS11), A Regulator for Tau Exon 10 Alternative Splicing Identified by an Expression Cloning Strategy,” Molecular and Cellular Biology 26:6739-6747 (2006), which is hereby incorporated by reference in its entirety). The key driver gene in the red module ENPP2 (FIG. 4C) was reported to be a novel CSF marker for neurodegeneration such as Alzheimer’s disease and Lewy Body Dementia (Heywood et al., “Identification of Novel CSF Biomarkers for Neurodegeneration and Their Validation by a High-Throughput Multiplexed Targeted Proteomic Assay,” Molecular Neurodegeneration 10:64 (2015), which is hereby incorporated by reference in its entirety), together with its neighbor node PSEN1 , suggesting common regulatory features between AD and PD. The 6^th ranked green-yellow module is primarily regulated by the epigenetic regulator BMI1. Bmil depletion was found to significantly delay retinal degeneration via CDK inactivation (Zencak et al., “Retinal Degeneration Depends on Bmi 1 Function and Reactivation of Cell Cycle Proteins,” Proceedings of the National Academy of Sciences of the United States of America 110:E593-601 (2013), which is hereby incorporated by reference in its entirety). The upregulation of BMI1 in PD might account for neuronal death. The important downstream of BMI1 includes DNAJB6, CD2BP2 and CIRBP. CD2BP2 and CIRBP were involved in miRNA regulation (Wang et al., “TEG-1 CD2BP2 Controls miRNA Levels by Regulating miRISC Stability in C. elegans and Human Cells,” Nucleic Acids Research 45:1488-1500 (2017), which is hereby incorporated by reference in its entirety) and H2O2-mediated cortical neuronal death (Li et al., “Cold-inducible RNA Binding Protein Inhibits H(2)O(2)-Induced Apoptosis in Rat Cortical Neurons,” Brain Research 1441:47-52 (2012), which is hereby incorporated by reference in its entirety) while DNAJB6 was accumulated in Lewy Body and was upregulated in PD (Durrenberger et al., “DnaJB6 is Present in the Core of Lewy Bodies and is Highly Up-Regulated in Parkinsonian Astrocytes,” Journal of Neuroscience Research 87:238-245 (2009), which is hereby incorporated by reference in its entirety).

[00120] The multiscale network depicts the global landscape of PD pathogenesis with a high resolution. To validate the network based findings, it was decided to focus on the top- ranked key driver gene STMN2 in the top ranked turquoise module which was most significantly enriched with DEGs in PD, contained multiple differentially expressed PD GWAS hits, showed reduced MDC and represented neuron-specific gene expression. This integrative network analysis suggested that: 1) the expression of STMN2 was down-regulated in PD cases compared to control (FIG. 8); 2) STMN2 was ranked the top among 52 key driver genes in the turquoise module and 3) The first-layer neighborhood of STMN2 implicated a potential role of STMN2 in synaptic vesicle trafficking (FIG. 8), which was significantly impaired in PD and indicated by the DEG analysis.

Example 6 - Knockdown of Stmn2 in midbrain impairs locomotor functions in mice. [00121] Since the analysis predicted Stmn2 as the top key driver gene in the sporadic PD network, a series of validation experiments were performed focused on Stmn2. As STMN2 is decreased in PD brains, it was sought to investigate the consequence of reduced Stmn2 expression in mouse models. See FIGS. 5A-5I and 11 A-l 1C. Abundant expression of Stmn2 was found in dopaminergic neurons (tyrosine hydroxylase positive, TH+) in substantia nigra of adult mouse brain (FIG. 9). Next, the knockdown efficiency of a RFP -tagged STMN2-shRNA construct in mouse neuroblastoma N2A cell line was validated by Western blot analysis and in primary midbrain neuron cultures by immunocytochemistry. Both methods demonstrated a -70% knockdown efficiency (FIG. 10). The validated Stmn2 shRNA sequence was then packaged into AAV and confirmed the infection and knockdown efficiency in N2A cells (FIG. 10). A unilateral injection of AAV2/1 carrying the Stmn2- targeting shRNA was performed into the substantia nigra to knock down Stmn2. The same serotype of AAV that carried a scrambled sequence was injected into different mice as control. The locomotor activity was examined by performing rotarod and open field tests (FIG. 5 A) in the fourth week after injection. The mice with Stmn2 -shRNA injection spent significantly shorter time on the rod than the control mice (FIG. 5B). Administration of amphetamine normally stimulates DA release and induces hyperactivity in mice, and it is commonly used for testing whether a lesion in locomotor activity is DA dependent. The amphetamine treatment induced a significant difference in rotational behavior in the two groups of injected mice: the control group displayed a preference to the ipsilateral side (FIG. 5C), whereas the Stmn2-shRNA group showed a preference to the contralateral side (FIG. 5D). In contrast, there was no difference in multiple behavioral features in open field between the two groups prior to amphetamine treatment, except that the Stmn2 -shRNA mice showed fewer vertical episode counts than control mice (p=0.038). Taken together, the data suggested that Stmn2 knockdown causes DA related locomotor function impairment.

Example 7 - Knockdown of Stmn2 in mice leads to DA neuron degeneration in substantia nigra and depletion of striatal DA content.

[00122] It was then asked if impaired locomotor activity is caused by loss of DA neurons by performing stereological counting of TH+ neurons of the substantia nigra in the injected mice. A striking (-75%) reduction of TH+ neurons was found in the mice with Stmn2 -shRNA injection compared to the mice with scrambled shRNA injection (FIGS. 6A and 6B). Increased cleaved caspase-3 immunostaining was found only in the Stmn2 -shRNA injected substantia nigra (FIG. 6C), confirming that the reduction of TH+ cell number was due to apoptotic cell death.

[00123] Next, striatal DA content was examined by performing High Performance Liquid Chromatography (HPLC). Indeed, a 50% reduction of DA as well as the three major metabolites (DOPAC, 3-MT and HVA), was observed in the ipsilateral (right) striatum compared to the contralateral (left) side in the mice with Stmn2 shRNA injection (FIG. 6D). No difference of DA and the metabolites was found between ipsilateral and contralateral sides in the mice injected with scrambled shRNA, indicating that the surgical impact was negligible. The density of dopamine transporter positive (DAT+) terminals in the striatum was examined based on DAT fluorescence intensity. A reduction of DAT+ terminal density was observed in the ipsilateral striatum of the mice with Stmn2 shRNA injection compared to that in the mice with scrambled shRNA injection, suggesting the knockdown of Stmn2 leads to the degeneration of DA neuron terminals (FIG. 6E). The above evidence is consistent with the observation that Stmn2 knockdown impaired locomotor activity and led to loss of TH+ neurons.

Example 8 - Stmn2 knockdown causes presynaptic dysfunction in primary DA neurons. [00124] The multiscale network analysis suggests that Stmn2 is connected to synaptic transmission and could be a key regulator of presynaptic activity (FIGS. 4A and 8). To elucidate the cellular mechanism underlying the protective function of Stmn2 in DA neurons (FIGS. 5A-5I and 6A-6G), an imaging assay was employed utilizing pHluorin for testing presynaptic function of cultured DA neurons. PHluorin is a variant of green fluorescence protein (GFP) whose fluorescence is quenched by protonation. When targeted to the acidic lumen of synaptic vesicles, pHluorin is quenched but fluoresces upon exocytosis when pHluorin is exposed to the extracellular buffer (pH 7.4). Sankaranarayanan et al., “Calcium Accelerates Endocytosis of vSNAREs at Hippocampal Synapses,” Nature Neuroscience 4:129-136 (2001), which is hereby incorporated by reference in its entirety. Conjugating pHluorin to vesicular transporters, such as vesicular glutamate transporter 1 (vGLUTl) or vesicular monoamine transporter-2 (vMAT2) was used to examine synaptic vesicle kinetics in a quantitative manner. Ariel et al., “Optical Mapping of Release Properties in Synapses,” Frontiers in Neural Circuits 4:18 (2010) and Pan et al., “Calbindin Controls Release Probability in Ventral Tegmental Area Dopamine Neurons,” Nat. Neurosci. 15:813-815 (2012), both of which are hereby incorporated by reference in their entirety. pHluorin imaging was performed on cultured DA neurons expressing vMAT2-pHluorin at 9-11 days after Stmn2 -shRNA transfection. While no morphological abnormality was observed in the Stmn2 -shRNA treated DA neurons, as compared to scrambled shRNA treated neurons, Stmn2 -shRNA treated DA neurons clearly display slower SV endocytosis as well as exocytosis than scrambled shRNA treated neurons (FIGS. 7A-7C), indicating that reduced Stmn2 expression results in impaired presynaptic transmission in DA neurons. The result suggests that disruption of SV trafficking caused by STMN2 deficiency may precede neuronal death as observed in vivo (FIGS. 6A-6G). The data thus corroborates the network analysis and points to a key role of STMN2 in regulating synaptic activity as shown in network analysis (FIGS. 4 A and 8).

[00125] Differing from many monogenic neurodegenerative diseases such as Huntington’s disease (HD), PD pathogenesis involves multiple genes and results from complex interactions of genetic and environmental factors as well as aging. Over the past two decades, the majority of PD research has focused on individual genes or pathways and lacked a systematic method capable of identifying relevant molecules or pathways underlying idiopathic PD pathogenesis. Furthermore, GW AS approaches have revealed many risk loci for PD; however, these loci have explained very little of the disease etiology and paths to treatment and prevention remain unclear. In contrast, systems/network biology based upon large genetic and genomic data for other complex diseases such as cancer, Alzheimer’s disease, obesity and diabetes has led to the discovery of novel mechanisms and disease target genes. These studies have revolutionized the research for complex diseases involving multiple layers of factors, including genetic, epigenetic, and environmental. Bailey et al., “Genomic Analyses Identify Molecular Subtypes of Pancreatic Cancer,” Nature 531:47-52 (2016); Chen et al., “Variations in DNA Elucidate Molecular Networks That Cause Disease,” Nature 452:429-435 (2008); Emilsson et al., “Genetics of Gene Expression and Its Effect on Disease,” Nature 452:423-428 (2008); Gadaleta et al., “Integration of Gene Expression and Methylation to Unravel Biological Networks in Glioblastoma Patients,” Genet. Epidemiol. 41:136-144 (2017); Vishnubalaji et al., “Genome-Wide mRNA and miRNA Expression Profiling Reveal Multiple Regulatory Networks in Colorectal Cancer,” Cell Death Dis. 6:el614 (2015); Zhang et al., “Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease,” Cell 153:707-720 (2013); Zhang et al., “A Network Medicine Approach to Build a Comprehensive Atlas for the Prognosis of Human Cancer,” Brief Bioinform. 17:1044-1059 (2016); Zhu et al., “Increasing the Power to Detect Causal Associations by Combining Genotypic and Expression Data in Segregating Populations,” PLoS Comput. Biol. 3:e69 (2007); and Zhu et al., “Integrating Large-Scale Functional Genomic Data to Dissect the Complexity of Yeast Regulatory Networks,” Nature Genetics 40:854-861 (2008), all of which are hereby incorporated by reference in their entirety. However, the lack of large-scale molecular data from PD post-mortem brains has impeded the application of such a systems approach in PD research. In addition, although many gene expression studies of PD have been reported, the scale of these studies is small and each alone failed to provide sufficient power to derive a holistic picture of PD related pathways or key regulators. To address this unmet challenge in PD research, gene expression data was assembled in human brains from 12 studies of PD into the largest gene expression dataset in PD research, thus far. This allowed the multiscale gene network analysis to systematically uncover intrinsic network structures and key causal regulators of PD. Network modules most enriched for the molecular changes in PD are involved in synaptic transmission and oxidative phosphorylation, mitochondrion, myelination and response to unfolded protein. Analysis of these modules and the causal network in PD further identified a number of key causal regulators including STMN2, which has an overall reduced expression in PD brains. This animal model studies suggest that Stmn2 deficiency is causative to dysfunctional synaptic transmission and neuronal viability in DA neurons underlying a common pathogenic mechanism for idiopathic PD.

[00126] STMN2 has never previously been implicated in PD pathogenesis. STMN2 , also known as superior cervical ganglion 10 (SCG10), is a ~20kDa phospho-protein of the stathmin family that destabilizes microtubules. Antonsson et al., “Identification of In Vitro Phosphorylation Sites in the Growth Cone Protein SCG10. Effect Of Phosphorylation Site Mutants on Microtubule-Destabilizing Activity,” The Journal of Biological Chemistry 273:8439-8446 (1998), which is hereby incorporated by reference in its entirety). STMN2 promotes neurite outgrowth (Xu et al., “Regulation of Neurite Outgrowth by Interactions Between the Scaffolding Protein, INK -Associated Leucine Zipper Protein, and Neuronal Growth-Associated Protein Superior Cervical Ganglia Clone 10,” The Journal of Biological Chemistry 285:3548-3553 (2010), which is hereby incorporated by reference in its entirety), axon formation (Li et al., “Rndl Regulates Axon Extension by Enhancing the Microtubule Destabilizing Activity of SCG10,” The Journal of Biological Chemistry 284:363-371 (2009), which is hereby incorporated by reference in its entirety) and regeneration after injury (Shin et al., “SCG10 is a JNK Target in the Axonal Degeneration Pathway,” Proceedings of the National Academy of Sciences of the United States of America 109:E3696-3705 (2012), which is hereby incorporated by reference in its entirety). In a chemical-induced PD mouse model, 4-week administration of MPTP downregulated Stmn2 level and the rescue of MPTP -induced phenotype restored Stmn2 expression. Sconce et al., “Intervention with 7,8-dihydroxyflavone Blocks Further Striatal Terminal Loss and Restores Motor Deficits in a Progressive Mouse Model of Parkinson’s disease,” Neuroscience 290:454-471 (2015), which is hereby incorporated by reference in its entirety. Here it is shown that Stmn2 is abundantly expressed in midbrain DA neurons (FIG. 9) and genetic manipulation to reduce Stmn2 levels in midbrain is sufficient to cause remarkable degeneration of DA neurons in the substantia nigra, depletion of striatal DA levels and DA related locomotor function deficits. Furthermore, it was shown that reduced STMN2 expression in midbrain DA neurons disrupted SV endo- and exo-cytosis, which is consistent with the previous reports that reduced STMN2 expression impairs protein secretion in PC12 cells (Mahapatra et al., “The Trans-Golgi Proteins SCLIP and SCG10 Interact With Chromogranin A to Regulate Neuroendocrine Secretion,” Biochemistry 47:7167-7178 (2008), which is hereby incorporated by reference in its entirety) and the overexpression of STMN2 facilitates APP clearance by promoting its trafficking to the cell surface (Wang et al., “SCG10 Promotes Non-Amyloidogenic Processing of Amyloid Precursor Protein by Facilitating its Trafficking to the Cell Surface,” Human Molecular Genetics 22:4888-4900 (2013), which is hereby incorporated by reference in its entirety). Thus it is hypothesized that the loss of STMN2 may contribute to PD pathogenesis via disruption of cytoskeleton dynamics and dysregulation of vesicle trafficking pathways. The exact mechanism whereby depletion of Stmn2 leads to DA neuron degeneration in vivo remains to be elucidated in the future. This experimental data, therefore, suggests that the endogenous expression levels of STMN2 determines the vulnerability of DA neurons and reduced STMN2 expression is causal to DA neuron degeneration and disease progression as part of pathogenic mechanism for the common PD.

[00127] Accordingly, rescue of an individual from a neurodegenerative phenotype is accomplished by administration to the individual of a therapeutically effective amount of an agent capable of enhancing the expression or activity or both of STMN2.

Example 9 - In vivo rescue experiments/Stmn2 overexpression system.

[00128] A Synapsin_Stmn2_IRES__GFP plasmid that overexpresses mouse Stmn2 under control of neuron-specific synapsin promoter was designed and constructed. STMN2 in human has two isoforms ENST00000220876 and ENST00000518111. See FIGS. 12 and 13. In Mount Sinai AD cohort study, ENST00000220876 was found to be the dominant isoform expressed (log2CPM around 9) in human brain and its expression is negatively correlated with clinical dementia rating (CDR) in BM36 and BM22 while ENST00000518111 is barely detected (log2CPM around 0).

[00129] The mouse Stmn2 gene only encodes one transcript (Accession No. NM_025285.2 (SEQ ID NO: 1) → Accession No. NP_079561.1 (SEQ ID NO: 2) stathmin-2, ENSMUSP00000029002) and the protein is identical to human isoform 2 (Accession No. NM_007029.3 (SEQ ID NO: 5) → Accession No. NP_008960.2 (SEQ ID NO: 6), ENSP00000220876) on the amino acid level, which has been cloned into the above plasmid.

The human STMN2 gene encodes two isoforms: isoform 1 (Accession No.

NM_001199214.1 (SEQ ID NO: 3) → Accession No. NP_001186143.1 (SEQ ID NO:

4), ENST00000518111) is a longer transcript but has low expression. Isoform 2 uses an alternative exon and has a distinct C terminal, which is more abundantly expressed according to the RNA sequencing results from Mount Sinai Brain Bank (Synapse: 3159438). The Synapsin_Stmn2_IRES__GFP is packaged into AAV9 for overexpression in the mouse brain by Vigene Biosciences (Rockville, MD) with titer around 10¹³ gc/ml. The overexpression of Stmn2 is examined prior to the in vivo injection.

Example 10 - Human wild type a-syn overexpression mouse model.

[00130] AAV9 that carries either Synapsin _GFP or Synapsin SNCA were injected into substantia nigra and behavioral and pathological tests were performed at 2, 4, and 10 weeks after injection. PD-like behavioral abnormality started to appear at 4 weeks after injection followed by detection of DA neuronal loss.

[00131] Two to four-month old C57bl/6j mice are divided into treatment groups and then injected with an AAV9 that carries one of the following: GFP only; Synapsin_Stmn2_IRES__GFP; human wild type a-syn; and human wild type a- syn+Synapsin_Stmn2_IRES__GFP. Behavioral tests such as rotarod and open field are performed 2, 4 and 8 weeks after injection. Once behavioral changes are observed, the mice are sacrificed and brain slices taken for pathological examination.

[00132] It was also shown that Stmn2 knockdown increased phosphorylation at Serl29 of a-syn Wild type, A30P and A53T a-synclein was induced by 1 μg/ml Dox in PC 12 cells after splitting cells into 6-well plates. 24 hours later, CMV-driven Stmn2 overexpression plasmids were transfected into these cells with increasing dose (with GFP plasmid: 1 : 1, 2: 1, and 4:1 ratio, making the total amount of DNA the same among groups). 48 hours after transfection, cells were collected and cell lysates were subjected to western blot. The results are shown in FIG. 14. [00133] Mouse Stmn2 sequence includes: NM_025285.2 (SEQ ID NO: 1) → NP_079561.1 (SEQ ID NO: 2) ENSMUSP00000029002) (transcript). A portion of SEQ ID NO: 1 is shown below (the shRNA targeting region is bolded).

[00134] Human isoform 2 sequence includes NM 007029.3 (SEQ ID NO: 5) → NP_008960.2 (SEQ ID NO: 6) , ENSP00000220876 (transcript). A portion of SEQ ID NO: 5 is shown below (the shRNA targeting region is bolded).

[00135] With overexpression of Stmn2 in these cells, significant changes in total a-syn protein level were not observed. However, there seemed to be a trend of increasing pS129 of a- syn.

Example 11 - snRNA Sequencing.

[00136] Single-nucleus RNA sequencing was performed in 33 postmortem substantia nigra tissue samples, including 9 healthy controls and 24 PD cases. After QC process, two samples were excluded due to poor data quality. Firs, the Seurat pipeline (Butler et al., “Integrating Single-Cell Transcriptomic Data Across Different Conditions, Technologies, and Species,” Nat. Biotechnol. 36(5):411-420 (2018) and Satija et al., “Spatial Reconstruction of Single-Cell Gene Expression Data,” Nat. Biotechnol. 33(5):495-502 (2015), both of which are hereby incorporated by reference in their entirety) was used for clustering analysis. Also identified were differentially expressed genes between controls and PD cases within each cluster. STMN2, which was downregulated in the postmortem PD brains by bulk-tissue microarray analysis (Wang et al., “The Landscape of Multiscale Transcriptomic Networks and Key Regulators in Parkinson ’s Disease, ” Nat. Commun. 10(1):5234 (2019), which is hereby incorporated by reference in its entirety), was also significantly down-regulated in PD in four cell clusters at the single-cell level, with p-values = 1.6E-05, 2.6E-12, 1.7E-07 and 2.4E-07, respectively. The single-nucleus RNA sequencing data confirm the down-regulation of STMN2 in neurons in PD and exclude the possibility that the observed down-regulation of STMN2 in the bulk tissue data is due to the loss of dopaminergic neurons.

Example 12 - Efficacy Study using Thy-1 Alpha Synuclein Mice.

[00137] Male Thy-1 alpha synuclein (Thy-1 α-Syn) mice (Line 61) (see Chesselet et al., “A Progressive Mouse Model of Parkinson’s Disease: The Thyl-aSyn (“Line 61”) Mice,” Neurotherapeutics 9:297-314 (2012), which is hereby incorporated by reference in its entirety) were bred at PsychoGenics. Mice were assigned unique identification numbers (ear notched) and housed in OptiMICE cages in groups of 3-4 animals. All animals were examined, manipulated, and weighed prior to initiation of the study to assure adequate health and suitability and to minimize non-specific stress associated with manipulation.

[00138] During the course of the study, 12/12 light/dark cycles are maintained. The room temperature is maintained between 20 and 23°C with a relative humidity maintained around 50%. Chow and water are provided ad libitum for the duration of the study.

[00139] Animals were single housed after surgery. Wet food was placed at the bottom of the cage for 3 days post-surgery. Treatment groups are described in Table 3.

-

[00140] Open Field Test - The open field test (“OF”) was used to assess both anxiety-like behavior and motor activity. The open field chambers are plexiglas square chambers (27.3 x 27.3 x 20.3 cm; Med Associates Incs., St Albans, VT) surrounded by infrared photobeam sources (16 x 16 x 16). The enclosure is configured to split the open field into a center and periphery zone and the photocell beams were set to measure activity in the center and in the periphery of the OF chambers. Animals having higher levels of anxiety or lower levels of activity tend to stay in the corners of the OF enclosures. On the other hand, mice that have high levels of activity and low levels of anxiety tend to spend more time in the center of the enclosure. Horizontal activity (distance traveled) and vertical activity (rearing) are measured from consecutive beam breaks. Animals were placed in the OF chambers for 30 minutes. Total ambulatory distance, ambulatory distance in center, total rearing, rate of rears in the center, and movement velocity were measured.

[00141] In the open field test, the striatal injection of AAV9 that carries Synaspin-Stmn2- IRES-EGFP into heterozygous Thy-1 α-synuclein mice (Line 61) led to significant increases in the Center Distance Traveled and Center Rearing Frequency compared to the sham-treated ones at the age of 14 weeks old. No significant changes in the total Distance Traveled or in the total Rearing Frequency was observed. Results of the open field test are shown in FIGS. 15A-15D. [00142] Changes in these parameters, Center Distance Traveled and Center Rearing Frequency, are usually associated with anxiety. Given the preliminary data, it is not conclusive but indicative that Stmn2 overexpression in the striatum (and maybe in the substantia nigra as well due to retrograde transport of the viral particles in the dopaminergic terminals, which needs confirmation by immunohistochemistry) could have some impact on the anxiety-related behaviors.

[00143] While the novel technology has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the novel technology are desired to be protected. As well, while the novel technology was illustrated using specific examples, theoretical arguments, accounts, and illustrations, these illustrations and the accompanying discussion should by no means be interpreted as limiting the technology. All patents, patent applications, and references to texts, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety.

[00144] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. A method for treating a neurodegenerative disorder characterized by loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites in an individual diagnosed with or suspected of having a loss of dopaminergic neurons and/or presence of Lewy bodies and Lewy neurites comprising: administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2.

2. The method of claim 1, wherein said neurodegenerative disorder is Parkinson’s Disease (PD).

3. The method of claim 1 or 2, wherein the agent is administered to the central nervous system of the individual.

4. The method of claim 1 or 2, wherein the agent is administered intrathecally, intranasally, intraperitoneally, orally, parenterally, nasally, subcutaneously, intravenously, intramuscularly, intracerebroventricularly, intraparenchymally, by intranasal inhalation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.

5. The method of any one of claims 1-5, wherein the agent capable of increasing the expression and/or activity of STMN2 is a vector comprising a nucleic acid encoding STMN2.

6. The method of claim 5, wherein the expression vector containing a nucleic acid encoding STMN2 is an adeno-associated viral vector (AAV).

7. The method of claim 6, wherein the AAV is AAV9.

8. The method of any one of claims 6-8, wherein the nucleic acid encoding STMN2 has the nucleotide sequence of SEQ ID NO: 5.

9. A method for the treatment of an individual with PD comprising administering to said individual a therapeutically effective amount of an agent capable of increasing the expression and/or activity of STMN2.

10. A vector comprising a nucleic acid encoding STMN2.

11. The vector of claim 10, wherein the vector is an adeno-associated viral vector (AAV).

12. The vector of claim 11, wherein the AAV is AAV9.

13. The vector of any one of claims 10-12, wherein said STMN2 is human.

14. The vector of any one of claims 10-12, wherein said STMN2 is murine.

15. The vector of claim 13, wherein the human STMN2 comprises the nucleotide sequence of SEQ ID NO: 5.

16. The vector of claim 14, wherein the murine STMN2 comprises the nucleotide sequence of SEQ ID NO: 1.