Use of neuronal nitric oxide synthase inhibitors in alzheimer's disease

Abstract

Disclosed are neuronal nitric oxide synthase (nNOS) inhibitors for use in methods for reducing AβO-induced tau phosphorylation, reducing AβO formation and accumulation on neurons, and modulating neuronal nitric oxide synthase (nNOS).

Description

USE OF NEURONAL NITRIC OXIDE SYNTHASE INHIBITORS IN ALZHEIMER’S DISEASECROSS-REFERENCE TO RELATED APPLICATIONSThis application claims benefit of priority to U.S. Patent Application Serial No. 63 / 514.798, filed July 20, 2023. The contents of which are incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under grant numbers GM105538 and GM131788 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUNDThe field of the invention relates to inhibitors of nitric oxide synthase for treatment of neurological diseases or disorders. More specifically, the invention relates to inhibitors of neuronal nitric oxide synthase for treatment of Alzheimer’s disease.Alzheimer’s disease (AD) is the most common cause of dementia, which afflicts approximately 1 in 9 people aged 65 and older. The histopathological diagnosis of AD is based on the presence of amyloid P (AP) plaques and neurofibrillary tangles of hyperphosphorylated tau protein. Soluble oligomers of AP (ApOs) instigate neurodegeneration by instigating oxidative and nitrosative stress, triggering inflammation, impairing autophagic machinery, and, importantly, inducing phosphorylation of tau protein.The binding of extracellular ApOs to neuronal receptors triggers intracellular signaling cascades that result in aberrant post-translational modification of proteins. For example, aberrant cysteine nitrosylation of several proteins is observed in AD brains, causing the activation of multiple tau kinases and the inhibition of a key chaperone; these post-translational modifications may contribute to tau hyperphosphorylation, synapse damage, and further protein aggregation. Additionally, the binding of ApOs to receptors triggers intracellular tau hyperphosphorylation. Hyperphosphorylation alters the function of tau, thus, hyperphosphorylated tau mediates a substantial portion of downstream ApO-induced neurotoxicity. However, the mechanisms by which ApO binding triggers intracellular tau phosphorylation and cysteine nitrosylation have not been well elucidated. Accordingly, there is a need in the art for methods for targeting ApO-induced tau phosphorylation as well as other processes implicated by AD.SUMMARY Disclosed are methods for reducing or preventing AβO-induced tau phosphorylation, increasing synaptic spine density, and reducing or preventing AβO accumulation in the presence of Aβ monomer by contacting neurons with an effective amount of a neuronal nitric oxide synthase inhibitor. The neurons can be contacted with the neuronal nitric oxide synthase inhibitor in vitro or ex vivo. Further, neurons can be contacted with the neuronal nitric oxide synthase inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder such as Alzheimer’s Disease. The neuronal nitric oxide synthase inhibitor can be a compound of a Formula (I) or a salt or solvate thereof: l, C1-C6-alkoxy, halogen, or haloalkyl,represents a single, double, or triple bond; Y is substituted aryl or substituted heteroaryl, wherein Y is substituted at one or more ring positions with halogen or a substituent having a formula —Z—Ra; or Y has a formula — Z—Ra; Z is selected from C1-C6-alkyl, C2-C6-alkenyl, and C2-C6-alkynyl; Rais selected from amino, alkylamino, dialkylamino, or a 4-6 membered heterocycle which contains at least one nitrogen atom and which heterocycle is optionally substituted at one or more positions with alkyl, alkoxy, or halogen. In particular, the neuronal nitric oxide synthase inhibitor can be HD-3-86, .PTION OF THE FIGURES Figure 1. Mechanism of NMDAR and nNOS-dependent excitotoxicity. AβOs and glutamate activate the N-methyl-D-aspartate receptor (NMDAR), causing local calcium ion influx. Memantine is an NMDAR inhibitor. Because neuronal nitric oxide synthase (nNOS) isphysically linked to the NMDAR via postsynaptic density protein 95 (PSD-95), the local calcium influx enables calmodulin (CaM) to activate nNOS, which produces nitric oxide (NO) that contributes to excitotoxicity downstream.Figure 2. Mechanism of physiological NMDAR / nNOS signaling. Physiological levels of glutamate and possibly amyloid beta oligomers ApOs activate the NMDAR to trigger local calcium influx, which activates neuronal nitric oxide synthase (nNOS) via calmodulin (CaM) binding. Activated nNOS produces nitric oxide (NO), which activates soluble guanylate cyclase (sGC), triggering cyclic guanosine monophosphate (cGMP)-mediated downstream activation of protein kinase G (PKG) and cAMP response element-binding protein (CREB), which mediate synaptic plasticity.Figure 3. Potential mechanisms of nNOS-mediated neurodegeneration. Amyloid beta oligomers (ApOs) trigger calcium dysregulation in neurons, which could lead to neuronal nitric oxide synthase (nNOS) overactivation, which would generate excess nitric oxide (NO). Nitric oxide activates the tau kinases glycogen synthase kinase-3p (GSK-3P) and cyclin-dependent kinase 5 (Cdk-5). NO also activates the small G protein Dexrasl by S-nitrosylation, which leads to synapse loss. NO can inhibit the protein disulfide isomerase (PDI) by S-nitrosylation and can trigger zinc release, which can contribute to aggregation and ApO formation.Figure 4. Glutamate induces NO production. (A) Representative images of rat hippocampal cells incubated with DAF-FM DA and treated with conditioned media as a control or 100 μM L-glutamic acid and 10 μM D-serine. Cells were excited at 495 nm and imaged at 515 nm using the l Ox objective. (B) Reaction of DAF-FM with NO. (C) Immunofluorescent integrated fluorescent density in control and glutamate treated wells, normalized to intensity in control wells. Data were combined from n=3 experiments, n=2 wells each. Integrated fluorescent density had an increased trend of 541x in the L-glutamate treatment (p = 0. 13 due to high standard deviation).Figure 5. NOS inhibition prevents glutamate NO production. (A) Immunofluorescent integrated fluorescent density' from rat hippocampal neurons imaged following incubation with DAF-FM DA (80 μM) for 2 hours, then treated with conditioned media. 100 μM L-glutamic acid and 10 μM D-serine, or pre-incubated with 200 μM L-NAME before incubation with 100 μM L-glutamic acid and 10 μM D-serine. Signal was normalized to intensity in control wells. Images used for quantification were collected using a lOx microscope objective in live cells, 4 images per well were averaged, and data were combined from n=2 experiments (data also included in FIG. 4), n=2 wells each. Integrated fluorescent showed a trend toward reduction by L-NAME (100%, p = 0.4047 due to high standard deviation). (B) Representative images of rathippocampal cells treated as described in (A) and then labeled with p-III-tubulin antibody and DAPI and imaged at high magnification.Figure 6. Nitric oxide production in neurons. Rat hippocampal neurons were incubated with DAF-FM DA (80 μM) for 2 hours, then 100 μM L-glutamic acid and 10 μM D-serine, and finally labeled with P-III tubulin. Neurons were imaged at Cy5 and FITC ranges with an ImageXPress microscope using the lOx objective. (A) Neurons were labeled using P-III tubulin (TUBB3. red). (B) Merged signal from P-III tubulin (red) and NO (green) are shown. (C) A histogram of % DAF-FM DA signal found within neuronal (TUBB3) area. Median value 66% of NO w as produced in neurons.Figure 7. nNOS expression is varied throughout neuron culture. Images of neuronal cell culture labeled with anti-nNOS antibody (green) w ith DAPI (blue) (A) or without DAPI shown (B). Cells were imaged at 60x. Some cells exhibited high nNOS expression (orange arrows), while others had low expression levels (blue arrow). (C) A histogram was used to quantify expression levels of nNOS throughout the cell culture in terms of integrated fluorescent density of nNOS antibody divided by area. The minimum value was 8.75. the maximum was 79.8, and the median was 21.7. Data were collected from n = 31 regions of interest from a total of 9 images.Figure 8. nNOS inhibition blocks pTau (Thr205). (A) Representative images of neurons that were pre-treated with HD-3-86 (100 μM), then treated with 500 nM ApO for 4 h. Cells were stained for ApOs (NU4, green), tau phosphorylated at Thr205 (pThr205, red), and nuclei (DAPI, blue) and mounted on slides before imaging using a 20x objective. (B) Tau phosphorylation at Thr205 w as quantified based on integrated fluorescent densify of pThr205 antibody divided by number of nuclei per image (n=15-24 images per condition). Tau phosphorylation at Thr205 was significantly increased in the Apo condition (p = 0.0001) and significantly reduced in the ApO+HD-3-86 condition (108%, p<0.0001). (C) ApO puncta per micron along processes were quantified from n = 18-19 images per condition, collected using a 63x objective. The ApO condition demonstrated significantly more ApO puncta than F12 vehicle (p <0.0001), and ApO binding was not reduced in the ApO+HD-3-86 condition (ns).Figure 9. nNOS inhibition blocks pTau (Ser396). (A) Representative images of neurons that were pre-treated with HD-3-86 (100 μM) or vehicle, then treated with 500 nM ApO or vehicle for 6 h. Cells were stained for ApOs (NU2, green), tau phosphorylated at Ser396 (pSer396, red), and nuclei (DAPI, blue) and mounted on slides before imaging with a 20x objective. (B) Integrated fluorescent densify of pSer396 divided by number of nuclei were analyzed for 30 images in each condition. ApO-treated cells displayed significantly more pTauthan F12 vehicle-treated cells (p <0.0001). HD-3-86 pre-treated cells had significantly lower levels of pTau than ApO-treated cells (97%. p <0.0001).Figure 10. nNOS inhibition increased synaptic puncta. (A) Representative images of neurons pre-treated with HD-3-86 (100 μM), then treated with 500 nM ApO for 1 h. Cells were stained for filamentous actin (phalloidin, green) and nuclei (DAPI, blue) and mounted on slides before imaging with a 63x objective. (B) Analysis of integrated fluorescent density of phalloidin was conducted using n = 10 images per condition. HD-3-86 pre-treated cells demonstrated an increase in integrated fluorescent density (1.5x, p = 0.0499).Figure 11. nNOS reduces Ap-derived Apo buildup . (A) Representative images of cells pre-treated with HD-3-86 (100 μM, 30 min), then Ap monomer (500 nM, 30 min), labeled for dendrites (MAP2, red), ApOs (NU2, green), and nuclei (DAPI, blue). (B) Analysis of APO puncta per micron along dendrites were quantified in n = 30 images per condition. APO buildup was observed in the Ap monomer condition (p < 0.0001) and was significantly prevented by nNOS inhibition (54%, p = 0.0057). This same trend was observed in a replicate experiment in a separate cell culture.Figure 12. Activation of tau kinases. ApOs trigger N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx, which enables calmodulin (CaM) binding to activate neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO). This could cause S- nitrosylation of cyclin-dependent kinase 5 (Cdk5), a tau kinase, activating it to enable increased production of phosphorylated tau (pTau) and synaptic spine loss. Ap also causes increased formation of p25, which also activates Cdk5 from p35. NO production by nNOS could also activate glycogen synthase kinase-3p (GSK3P), another tau kinase. Dashed lines indicate hypothesized pathways.Figure 13. Potential Fyn contribution to nNOS signaling. Hyperphosphorylated tau (pTau) tethers the kinase Fyn to the post-synaptic density 95 (PSD-95) scaffold protein, where it can phosphorylate the N-methyl-D-aspartate (NMDA) receptor to stabilize the interaction of the NMD AR and PSD-95. This may enhance activation of neuronal nitric oxide synthase (nNOS) by calmodulin (CaM) when NMD AR activation causes local calcium influx.Figure 14. Tau acetylation. Ap stimulates calcium influx into neurons, which activates neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO) which could nitrosylate glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH acety lates p300, enabling it to acety late tau. By trans-nitrosylation, GAPDH also inhibits the tau deacetylase (sirtuin 1) SIRT1, reducing tau de-acetylation. Dashed lines indicate hypothesized pathways.Figure 15. CAPON and Dexrasl activation. Ap triggers increased interaction of the C- terminal PDZ ligand of nitric oxide synthase (CAPON) with neuronal nitric oxide synthase (nNOS). This interaction enables nitrosylation of Dexrasl. S-nitrosylation enables Dexrasl to damage mitochondria. Tau also binds to CAPON, and nNOS-CAPON interaction allows increased nitration of tau at Tyr29, promoting tau aggregation.Figure 16. nNOS antibody titration. Quantification of raw integrated fluorescent density from three dilutions of nNOS antibody (1 :250. 1 :500, 1 : 800) that were compared to a secondary only staining condition. nNOS signal was significantly different from secondary only at 1:500 (p = 0.0153). Image analysis was conducted in ImageJ and data were analyzed using one-way ANOVA with post-hoc Dunnett's T3 multiple comparisons test.Figure 17. AT8 optimization. (A) Using AT8, cells were treated with 500 nM xLABO, 6h. Analysis of integrated fluorescent density of AT8 signal divided by integrated fluorescent density of total tau was used to calculate tau phosphorylation (n = 29-30 images / condition from 3 CS). A modest, but significant increase in tau phosphorylation was detected (1.4x, p = 0.0006) In a follow-up experiment, cells were treated with 500 nM xLABO. 6h, and tau phosphorylation was measured using different antibody aliquots in (B) and (C), resulting in different outcomes as measured by integrated fluorescent density of AT8 signal divided by integrated fluorescent density7of total tau (n = 20 images / condition from 2 CS). No significant difference was observed in (B), but a significant increase upon treatment was observed using (C) (4.4x, p =0.0083).Figure 18. Dose-response for block of ApO-induced pTau by nNOS inhibitors in HT22 hippocampal nerve cell line. HD-3-86 (HD) was tested with AT8 and pThr217 antibodies.DETAILED DESCRIPTIONA direct role for neuronal nitric oxide synthase (nNOS) in amyloid beta oligomer (ApO)-related neurodegeneration mechanisms has not been previously established. The Examples disclosed herein demonstrate that multiple forms of AD neurodegeneration can be disrupted by application of nNOS inhibitors. Disclosed herein are strategies to block ApO- related neurodegeneration that include inhibition of nNOS. More specifically. nNOS inhibitors can be effective in reducing ApO-induced tau phosphorylation, APO formation and accumulation on neurons, and modulation of spine morphology. nNOS inhibitors as disclosed herein can be used as a research tool to investigate mechanisms of ApO-related neurodegeneration. As a research tool, the nNOS inhibitors can be used in vitro and ex vivo assays related to ApO-related neurodegeneration. Furthermore, the nNOS inhibitors asdisclosed herein can be used as a baseline comparison of efficacy in testing the efficacy of other nNOS inhibitors in ApO-related neurodegeneration mechanisms.A subject in need of a treatment for a neurodegenerative disease or disorder can be treated with an nNOS inhibitor. The neurodegenerative disease or disorder can be Alzheimer’s Disease. The subject may be in need of treatment for reducing or preventing ApO-induced tau phosphorylation, reducing ApO accumulation, or increasing synaptic spine density. nNOS inhibitors as disclosed herein may be used in companson testing of the efficacy of other nNOS inhibitors in these and other neurodegeneration mechanisms.Testing of the nNOS inhibitors in therapeutic applications can be used to investigate the effects of long-term administration. Furthermore, the nNOS inhibitors can be used in tests to delineate treatment conditions in which normal function of NOS is preserved, but neurodegeneration mechanisms are reduced or prevented.The present invention is described herein using several definitions, as set forth below and throughout the application.DefinitionsThe disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.As used in this specification and the claims, the singular forms “a,” “an,"’ and “the’' include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentiallyof should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.The phrase '‘such as” should be interpreted as ’‘for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone. C alone, A and B together, A and C together, B and C together, and / or A, B. and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1. 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”A “subject in need thereof’ as utilized herein refers to a subject in need of treatment for a disease or disorder associated with neuronal nitric oxide synthase (nNOS) activity and / or nitric oxide (NO) levels, such as a disease or disorder in which elevated NO levels are undesirable. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.Diseases and disorders associated with nNOS activity may include, but are not limited to neurological diseases and diseases and disorders. Neurological diseases and disorders may include, but are not limited to neurodegenerative diseases and disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, and amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain damage, and migraine headaches. The disclosed compounds may be utilized to modulate enzyme activities including, but not limited to nNOS activity. The term “modulate” should be interpreted broadly to include “inhibiting” enzyme activity and / or otherwise modulating enzyme activity. Chemical Entities Chemical entities may be disclosed herein and may be described using terms known in the art and defined herein. The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively. The term “alkylene” refers to a diradical of an alkyl group. An exemplary alkylene group is -CH2CH2-. The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen, for example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like. The term “heteroalkyl” as used herein refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxyl” group. The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively. A “cycloalkene” is a compound having a ring structure (e.g., of 3 or more carbon atoms) and comprising at least one double bond. The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively. The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl,alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted. The term “cycloalkylene” refers to a diradical of a cycloalkyl group. The term “partially unsaturated carbocyclyl” refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl is not aromatic. The partially unsaturated carbocyclyl may be characterized according to the number or ring carbon atoms. For example, the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively. The partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle, spirocyclic carbocycle, or other carbocyclic ring system. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that are partially unsaturated. Unless specified otherwise, partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted. The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, - C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions withhalogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.The terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using 5 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position.The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, tert-butoxy and the like.An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl. such as may be represented by one of -O-alkyl, -O-alkenyL -O-alkynyl, and the like.The term “carbonyl” as used herein refers to the radical -C(O)-.The term “carboxy” or "carboxyl" as used herein refers to the radical -COOH or its corresponding salts, e.g. -COONa. etc.The term “amide” or “amido” or "carboxamido" as used herein refers to a radical of the form -R1C(O)N(R2)-, -R1C(O)N(R2) R3-, -C(O)N R2R3, or -C(O)NH2, wherein R1, R2and R3are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, and, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl. heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. Thepresent invention encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.Pharmaceutical CompositionsThe compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that inhibits neuronal nitric oxide synthase may be administered as a single compound or in combination with another compound that inhibits neuronal nitric oxide synthase or that has a different pharmacological activity.As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds, which are substantially nontoxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases. Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, α-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like. The particular counter-ion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity. Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.In addition, the methods disclosed herein may be practiced using solvate forms of the compounds or salts, esters, and / or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.The pharmaceutical compositions may be utilized in methods of treating a disease or disorder associated with neuronal nitric oxide synthase activity. As used herein, the terms “treating'’ or “to treat" each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.As used herein the term “effective amount" refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a disease or disorder associated with neuronal nitric oxide synthase activity.An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.A typical daily dose may contain from about 0.01 mg / kg to about 100 mg / kg (such as from about 0.05 mg / kg to about 50 mg / kg and / or from about 0. 1 mg / kg to about 25 mg / kg) of each compound used in the present method of treatment.Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and / or about 25 mg. The term “unit dosage form" refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to producethe desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subj ect and the caregiver.As one skilled in the art will appreciate, suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration but not suitable for intracerebral administration.The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdermal patches, and suspensions. In general, compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. The amount of the compound, however, is best defined as the “effective amount”, that is, the amount of the compound which provides the desired dose to the patient in need of such treatment. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders.Tablets are prepared by direct compression, by wet granulation, or by dry’ granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (inaddition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.Tablets can be coated with sugar, e g., as a flavor enhancer and sealant. The compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty7that some patients experience in swallowing solid objects.A lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, com and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a fdm of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.Transdermal patches can also be used to deliver the compounds. Transdermal patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition, and which holds the resinous composition in contact with the skin. Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality of pores through w hich the drugs are pumped by osmotic action.As one skilled in the art will also appreciate, the formulation can be prepared with materials (e.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) havingproperties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and / or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.Neuronal nitric oxide synthase inhibitor compoundsThe neuronal nitric oxide synthase inhibitor as used herein can include an aminopyridine moiety, as disclosed in U.S. Patent Nos. 10,167,260; 8,932,842, 8,927,730, 8,618,143, 8,557,552, 8,299,100, 8,278,084, and 7,470,790; PCT publication WO / 2021 / 173111; and U.S. Patent Publication Nos. 2021 / 0269400 and 2020 / 0331910, which are incorporated herein in their entirety for all purposes. In some embodiments, the neuronal nitric oxide synthase inhibitor can include an aminoquinoline moiety, such as the inhibitors disclosed in U.S. Patent Nos. 9,783,500, 9,663,468, and 9,212,144, and PCT publication WO / 2021 / 081528, which are incorporated herein in their entirety for all purposes. In other embodiments, the neuronal nitric oxide synthase inhibitor can include a pyrimidine moiety, such as the inhibitors disclosed in U.S. Patent No. 9.878,996 and PCT Publication No. WO / 2017 / 214286, which are incorporated herein in their entirety for all purposes. In still further embodiments, the neuronal nitric oxide synthase inhibitor can be a peptidomimetic, such as the inhibitors disclosed in U.S. Patent Nos. 7,470,815, 6,274,557, and PCT Publication No. WO / 2003 / 000198, which are incorporated herein in their entirety for all purposes. In still further embodiments, the neuronal nitric oxide synthase inhibitor can include a thiophene moiety, such as the inhibitors disclosed in U.S. Patent No. 9,682,950, which is incorporated herein in its entirety for all purposes.In one example, the neuronal nitric oxide synthase inhibitor can be 2-aminopyridine derived compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds and pharmaceutical compositions for treating diseases and disorders associated with neuronal nitric oxide synthase activity'. The disclosed compounds may include derivatives of 2-aminopyridine compounds or salts thereof having a formula as follows:where X is hydrogen, Ci-Cs-alkyl (e.g., methyl), Ci-Ce-alkoxy (e.g., methoxy), halogen (e.g.. fluoro or chloro), or haloalkyl (e.g., CH2F, CF2H, or CFs),represents a single, double, or triple bond; Y i ubstituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted quinolinyl such as substituted quinolin-3-yl), wherein Y is substituted at one or more ring positions with halogen or a substituent having a formula -Z-Raand Y optionally is substituted at two or more ring positions with halo (e.g., 2,3-difluoro-phenyl); or Y has a formula -Z-Ra; Z is selected from C1-C6-alkyl, C2-C6-alkenyl, and C2-C6-alkynyl; Rais selected from amino, alkylamino (e.g., methylamino), dialkylamino (e.g., dimethylamino), or a 4-6 membered heterocycle which contains at least one nitrogen atom and which heterocycle is optionally substituted at one or more positions with alkyl (e.g., methyl), alkoxy (e.g., ethoxy), or halogen (e.g. fluoro). Also contemplated are salts of the disclosed compounds including pharmaceutically acceptable salts of the disclosed compounds. Also contemplated are solvates of the disclosed compounds. In some embodiments, the disclosed compounds may have Raselected from pyrrolidinyl (e.g., pyrrolidin-2-yl) which optionally is substituted at one or more positions with alkyl (e.g., 1-methyl-pyrrolidin-2-yl or 4-methyl-pyrrolidin-2-yl) or alkoxy (e.g., 4-ethoxy- pyrrolidin-2-yl) or halogen (e.g., 4-fluoro-pyrrolidin-2-yl) or both of alkyl and halogen (e.g., 4-fluoro-1-methylpyrrolidin-2-yl), azetinyl (e.g., azetin-2-yl), which optionally is substituted at one or more positions with alkyl (e.g., 1-methyl-azetin-2-yl), and morpholinyl (e.g., morpholin-3-yl) optionally substituted at one or more position with alkyl (e.g., 4-methyl- morpholin-3-yl), piperidinyl (e.g., piperidin-1-yl), or piperazinyl (e.g., piperazin-1-yl) optionally substituted at one or more positions with alkyl (e.g., 4-methyl-piperazin-1-yl). In some embodiments, the disclosed compounds may have a formula (Ia): X a).where X, Z, and Raare as defined for formula (I) and R2, R3, R4, and R6, are each independentlyH or halogen (e.g., fluoro). In some embodiments. R' is halogen (e.g., fluoro). In some embodiments, R2is halogen (e.g., fluoro). In some embodiments, R2is halogen (e.g., fluoro) and R3is halogen (e.g., fluoro). In some embodiments, Z is methyl, ethyl, or propyl. In some embodiments, Rais dimethylamino.Specifically, the disclosed compounds may have a formula (lb)where X is defined as above and particularly where X is methyl; and where Rais defined as above and particular where Rais methylamino, dimethyl amino.Specifically, the disclosed compounds may have a formula (II):In some embodiments, the disclosed compounds may have a formula (Ic):where Z and Ra are as defined for formula (I) and R2, R3, R4, and R6, are each independently H or halogen (e.g., fluoro).The compounds disclosed herein may include compounds having a formula selected from:where X is selected from Cl, F, OMe, CH2F, CHF2, and CF3.Additionally, or alternatively, the compounds disclosed herein may include compounds, or salts thereof, having a formula selected from:In particular, the neuronal nitric oxide synthase inhibitor can be 6-[2-[5-[2- (dimethylamino)ethyl]-2,3-difluorophenyl]ethyl]-4-methyl-2-pyridinamine, or a salt thereof. The compound has the structureThe compound may also be referred to as HD-3-86.Salts of any of the disclosed compounds are contemplated herein. In particular, pharmaceutically acceptable salts of the disclosed compounds are contemplated herein.The disclosed compounds, salts thereof, and / or hydrates thereof may be formulated as pharmaceutical compositions comprising the compounds, salts thereof, and / or hydrates thereof, in a pharmaceutically acceptable carrier. The pharmaceutical compositions may be formulated for treating diseases or disorders associated with nitric oxide synthase activity.In some embodiments, the disclosed compounds and pharmaceutical compositions maybe utilized for treating or preventing a subject having a disease or disorder associated with nitric oxide synthase (NOS), the method comprising administering to a subject the compounds and / or the pharmaceutical compositions. In these methods, the subject may be administered an amount of the compound sufficient to inhibit NOS activity.In some embodiments, the disclosed compounds and pharmaceutical compositions may be utilized for treating or preventing neurodegenerative diseases or disorders in a subject in need thereof. In these methods, the subject may be administered an amount of the compound sufficient to inhibit NOS activity-. In these methods, the subject may have a neurodegenerative disease or disorder selected from, but not limited to, Alzheimer's disease (e.g., mild cognitive impairment (MCI) due to Alzheimer's disease or Stage 1, or “mild". Alzheimer’s disease), Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cerebral palsy, and migraine headaches.In some embodiments, the disclosed compounds and pharmaceutical compositions maybe utilized for inhibiting NOS in a subject in need thereof. In these methods, the subject may be administered an amount of the compound sufficient to inhibit NOS activity.In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS. In some embodiments, the disclosed compounds have a Ki for human nNOS which is less than about of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM. 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM or lower.In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS which is inhibited selectively versus iNOS. In some embodiments, the compounds exhibita selectivity for nNOS versus iNOS (n / i) which is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or higher. In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS which is inhibited selectively versus eNOS. In some embodiments, the compounds exhibit a selectivity for nNOS versus eNOS (n / e) which is at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or higher. In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS present in the brain of a subject in need thereof. In these methods, the disclosed compounds and preferably exhibit an effective permeability (Pe) for the blood brain barrier of at least about 5 × 10-6cm / s, 6 × 10-6cm / s, 7 × 10-6cm / s, 8 × 10-6cm / s, 9 × 10-6cm / s, 10 × 10-6cm / s, 11 × 10-6cm / s, 12 × 10-6cm / s, 13 × 10-6cm / s, 14 × 10-6cm / s, 15 × 10-6cm / s, 16 × 10-6cm / s, 17 × 10-6cm / s, 18 × 10-6cm / s, 19 × 10-6cm / s, or 20 × 10-6cm / s. Disruption of tau phosphorylation by nNOS inhibition As shown in Example 1, nNOS inhibition can be used to reduce tau phosphorylation at Thr205 (Figure 8 B) and at Ser396 (Figure 9) in neurons exposed to AβO. Disclosed herein is a method for reducing or preventing AβO-induced tau phosphorylation. The method includes comprising contacting neurons with an effective amount of an nNOS inhibitor. In some embodiments, the method of contacting neurons with an effective amount of the nNOS inhibitor reduces or prevents AβO-induced tau phosphorylation at Thr205. In other embodiments the method of contacting neurons with an effective amount of the nNOS inhibitor reduces or prevents AβO-induced tau phosphorylation at Ser396. The method as disclosed herein can include contacting the neurons with the nNOS inhibitor in vitro for reducing or preventing AβO-induced tau phosphorylation at Thr205 or Ser396. In some embodiments, the method can include contacting the neurons with the nNOS inhibitor ex vivo to reduce or prevent AβO-induced tau phosphorylation at Thr205 or Ser396. In some embodiments, the method includes contacting the neurons with the nNOS inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder. In some embodiments, the neurodegenerative disease or disorder is optionally Alzheimer’s Disease. In other embodiments, the subject is in need of a treatment for reducing or preventing AβO- induced tau phosphorylation. Disruption of amyloid beta oligomer (AβO) accumulation by nNOS inhibition The buildup of AβO along neurons is a hallmark of AD. As shown in Example 1, nNOS inhibition can reduce Aβ-derived AβO buildup as measured by puncta per micron along dendrites as shown in Figure 11. Disclosed herein is a method for disrupting AβOaccumulation. The method includes contacting neurons in vitro or ex vivo with an effective amount of a neuronal nitric oxide synthase inhibitor in the presence of Ap monomer.In some embodiments, the method includes contacting the neurons with the nNOS inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder. In some embodiments, the neurodegenerative disease or disorder is optionally Alzheimer’s Disease. In other embodiments, the subject is in need of a treatment for increasing synaptic spine density. Increasing synaptic spine density by nNOS inhibitionIn AD, the degeneration of synaptic spines correlates closely with memory loss. As shown in Example 1, nNOS inhibition can protect and enhance synapses. In one example, treatment of neurons with an nNOS inhibitor induced an increase in synaptic spine density (Figure 10). The method as disclosed herein can include contacting the neurons with the nNOS inhibitor in vivo to increase in synaptic spine density. In some embodiments, the method can include contacting the neurons with the nNOS inhibitor ex vivo to increase in synaptic spine density.In some embodiments, the method includes contacting the neurons with the nNOS inhibitor in a subj ect in need of a treatment for a neurodegenerative disease or disorder. In some embodiments, the neurodegenerative disease or disorder is optionally Alzheimer’s Disease. In other embodiments, the subject is in need of a treatment for increasing synaptic spine density. Use of nNOS inhibitors in cell-based assaysAdditionally, and alternatively as disclosed herein. nNOS inhibitors can be used in cellbased assays to investigate nNOS-dependent neurodegeneration, and particularly neurodegeneration related to AD. The nNOS-dependent neurodegeneration can be related to at least one of the mechanisms of ApO-induced tau phosphorylation, ApO formation, and modulation of spine morphology. For example, nNOS inhibitors as disclosed herein can be used in vitro to ascertain the effects of nNOS inhibitors on ApO-induced tau phosphorylation, in particular phosphorylation at Thr205 and pSer396. In another example, nNOS inhibitors can be used in vitro to ascertain the effects of nNOS inhibitors on ApO formation or accumulation of ApO at neurons. Additionally, nNOS inhibitors as disclosed herein can be used in vitro to ascertain the effects of nNOS inhibitors on synaptic spine density. Further, nNOS inhibitors as disclosed herein can be used ex vivo to ascertain the effects of nNOS inhibitors on at least one of the mechanisms of ApO-induced tau phosphory lation, ApO formation and accumulation, and modulation of spine morphology.In some embodiments, the nNOS inhibitors can be used to investigate neurodegeneration mechanisms in vitro or ex vivo for the purposes of comparing the effects ofother nNOS inhibitors on the mechanism. In other embodiments, NOS inhibitors can be used to investigate neurodegeneration mechanisms in vitro or ex vivo for the purposes of comparing the effects of other NOS inhibitors on the mechanism.In some embodiments, the nNOS inhibitors can be used to investigate effects of longterm administration therapeutic application of nNOS inhibitors. In other embodiments, the nNOS inhibitors can be used to identify an appropriate therapeutic window, or range of treatment conditions such as dosage amount, dosage, in which normal function of NOS is preserved, and aberrant signaling is prevented.EXAMPLESThe following Examples are illustrative and are not intended to limit the scope of the claimed subject matter.Example 1ApOs trigger tau phosphorylation and neurodegenerationThe histopathological diagnosis of AD is based on the presence of amyloid P (AP) plaques and neurofibrillary tangles of hyperphosphorylated tau protein2. However, the presence of Ap plaques does not ensure AD development, and the density of Ap plaques does not correlate well with cognitive decline3. Rather, soluble oligomers of Ap (ApOs) instigate neurodegeneration4'13. Apo levels can be used to detect AD in very early stages and correlate with cognitive impairment throughout disease14'18. ApOs cause neurodegeneration in multiple ways, including by instigating oxidative and nitrosative stress, triggering inflammation, impairing autophagic machinery, and, importantly, inducing phosphorylation of tau protein13-19'22.The binding of extracellular ApOs to neuronal receptors triggers intracellular signaling cascades that result in aberrant post-translational modification of proteins23,24. While these modifications are typically important for modulating protein activity to maintain homeostasis, aberrant post-translational modifications can lead to cellular dysfunction. For example, aberrant cysteine nitrosylation of several proteins is observed in AD brains, causing the activation of multiple tau kinases and the inhibition of a key chaperone; these post-translational modifications may contribute to tau hyperphosphorylation, synapse damage, and further protein aggregation19,20. Additionally, the binding of ApOs to receptors triggers intracellular tau hyperphosphorylation25,26. Hyperphosphorylation alters the function of tau, leading to mislocalization, aberrant protein interactions, and eventual formation of the characteristic neurofibrillary tangles26'30. Thus, hyperphosphorylated tau mediates a substantial portion of downstream ApO-induced neurotoxicity31'33. However, the mechanisms by which APObinding triggers intracellular tau phosphory lation and cysteine nitrosylation have not been well elucidated.Connection between nNOS and AfO bindingMechanistically, ApOs are known to activate the N-methyl-D-aspartate receptor (NMD AR), a ligand-gated calcium channel (FIG. I)34'36. One of four drugs currently used to treat AD, memantine, is an NMD AR antagonist that inhibits acute downstream reactive oxygen species formation and calcium influx following exposure to ApOs37. Throughout the course of AD, in addition to NMD AR activation, dysregulation of other calcium channels, such as the ryanodine receptor, also contribute to increasing intraneuronal calcium in AD38,39. The excitotoxicity following ApO-induced calcium influx could be mediated by neuronal nitric oxide synthase (nNOS). nNOS is physically linked to the NMDAR via postsynaptic density protein 95 (PSD-95)40,41. When the NMDAR is activated, the subsequent local calcium influx enables calmodulin to activate nNOS42'45.Physiologic and pathologic roles of nNOSActivation of nNOS causes increased production of nitric oxide (NO), which under normal conditions allows a functional response: activation of soluble guanylate cyclase, triggering cyclic guanosine monophosphate (cGMP)-mediated downstream activation of protein kinase G and cAMP response element-binding protein (FIG. 2)46'49. These processes underlie the normal signaling functions of nNOS, which include long-term potentiation and blood vessel dilation50. In early AD. increased activation of nNOS has been proposed to provide a compensatory mechanism by which neurons enhance synaptic signaling38. However, over the long term, overactivation of nNOS mediates excitotoxicity by excess cysteine nitrosylation and tyrosine nitration of protein targets and by contributing to nitrosative stress19-51. For example, in a mouse model of glutamate-induced excitotoxicity, disruption of the PSD-95 / nNOS interaction rescued neurons from cell death52,53. Additionally, anonselective inhibitor that targeted multiple isoforms of nitric oxide synthase (NOS) and NMDAR activity was also neuroprotective against glutamate-induced excitotoxicity54. Overall, these results point to nNOS as a mediator of neurodegeneration downstream of NMDAR activation and dysregulated calcium influx. However, it is currently unclear whether nNOS activation overall plays a neuroprotective or neurodegenerative role in ApO-induced AD pathology.Evidence for the overactivation of nNOS in ADThe loss of nNOS-expressing neurons in the hippocampus of AD patients suggests that nNOS overactivation may contribute to AD neurodegeneration55,56. Evidence for nNOS overactivation in the AD brain includes excess nitrosylation of cysteines20,57'59. Althoughnitrosylation of cysteine targets serves a functional role at physiological levels, overactivation of this pathway has been implicated in neuronal dysfunction. Also increased in the AD brain is the chemically distinct, NO-dependent nitration of protein tyrosine residues60. Work by Luth and coworkers suggests that nNOS is responsible for tyrosine nitration in neurons, while other isoforms of NOS including inducible NOS (iNOS) and endothelial NOS (eNOS), produce the majority of NO in glial cells61. However, iNOS is capable of producing higher levels of NO, which may diffuse from where it is produced in glial cells to have downstream consequences in neurons50. As such, the relative contribution of nNOS to NO-dependent changes in the AD brain remains unknown.Potential targets of nNOS in ADSeveral targets altered by nitric oxide in AD could be triggered by nNOS overactivation (FIG. 3). Two prominent tau kinases, glycogen synthase kinase-3(362and cyclin-dependent kinase 563, are both activated by nitric oxide. Additionally, activation of the small G protein Dexrasl by S-nitrosylation is implicated in ApO-induced synapse loss64’65. Further, S- nitrosylation of the chaperone protein disulfide isomerase is observed in AD, and the resultant inhibition of chaperone activity would be expected to accelerate protein aggregation66. Alternatively, nitric oxide also triggers zinc release, which can stabilize ApO formation67’68. By modulating these targets, nNOS overactivation could be involved in promoting ApO- induced tau phosphorylation, synaptic spine loss, and AD-relevant protein aggregation.The role of nNOS in contributing to these forms of AD-relevant neuronal damage has not been established. The exact role of the neuronal isoform of nitric oxide synthase has not been established in these pathways. Highly selective inhibitors of nNOS have not been previously applied to the study of AD, and as such, present a novel tool for the interrogation of this system. Therefore, in this work the inventors applied a specific inhibitor of nNOS for the first time to a model of ApO-induced tau phosphorylation to determine the effects of nNOS activity on ApO-induced tau phosphorylation, to measure whether nNOS activity contributed to ApO buildup from Ap monomer, and to test the effect of nNOS inhibition on synaptic puncta density.Mechanism, hypothesis and support for the role of nNOS in ApO-induced tauopathy.The mechanism of action for ApOs upstream from tau involves binding at PSD-95- labeled synapses, which stimulates NMDAR and the elevation of Ca2+and ROS. ApOs and glutamate activate the N-methyl-D-aspartate receptor (NMDAR), causing local calcium ion influx. nNOS is physically linked to the NMDAR via PSD-95, and the local calcium influx enables calmodulin (CaM) to activate nNOS, which produces nitric oxide that contributes topathogenic pTau downstream. Therefore, it was tested if nNOS, which is associated with PSD- 95 and NMDA receptors, might have a role in ApO-induced tauopathy. (See Figure 1). nNOS has not been investigated previously in regard to ApO-induced tauopathy but is implicated in AD.The inventors found the pTau response was inhibited robustly by HD-3-86, a highly selective nNOS inhibitor (Figure 8B, 9B). Results were obtained with hippocampal cell cultures, extensively used to investigate Apo toxicity. Results have been confirmed and extended with the HT-22 hippocampal nerve cell line. (Data not shown).Hippocampal cells were cultured according to the methods described in the following references, included in their entireties herein. (1) De Felice, F.G., Wu, D., Lambert, M.P., Fernandez. S.J., Velasco, P.T.. Lacor, P.N., Bigio. E.H.. Jerecic, J., Acton, P.J.. Shughrue, P.J. and Chen-Dodson, E., 2008. Alzheimer's disease-type neuronal tau hyperphosphorylation induced by Ap oligomers. Neurobiology of aging, 29(9), pp.1334-1347. (2) Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos. M., Morgan, T.E., Rozovsky,L, Trommer, B., Viola, K.L. and Wais, P., 1998. Diffusible, nonfibrillar ligands derived from Api-42 are potent central nervous system neurotoxins. Proceedings of the National Academy of Sciences, 95(11), pp.6448-6453. (3) Pitt, J., Wilcox, K.C., Tortelli, V., Diniz, L.P., Oliveira,M.S., Dobbins, C., Yu, X.W., Nandamuri, S., Gomes, F.C., DiNunno, N. and Viola, K.L., 2017.Neuroprotective astrocyte-derived insulin / insulin-like growth factor 1 stimulates endocytic processing and extracellular release of neuron-bound Ap oligomers. Molecular Biology of the Cell, 28(20), pp.2623-2636. (4) He M, Liu J, Cheng S, Xing Y, Suo WZ. Differentiation renders susceptibility to excitotoxicity in HT22 neurons. Neural Regen Res. 2013 May 15;8(14):1297-306. doi: 10.3969 / j.issn. 1673-5374.2013. 14.006. PMID: 25206424; PMCID: PMC4107644. (5) Poejo, Joana, Yolanda Orantos-Aguilera, Francisco Javier Martin-Romero, Ana Maria Mata, and Carlos Gutierrez-Merino. "Internalized amyloid-P (1-42) peptide inhibits the store-operated calcium entry in HT-22 cells." International Journal of Molecular Sciences 23, no. 20 (2022): 12678. (6) Lim, J., Bang, Y„ Kim, K.M. and Choi, H.J., 2023. Differentiated HT22 cells as a novel model for in vitro screening of serotonin reuptake inhibitors. Frontiers in Pharmacology, 13. p. 1062650. (7) www.sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 300 / 433 / 20267562. pdf.A series of reproducible dose curves using pTau antibodies AT8 and pThr217 are shown in Figure 18 for HD-3-86 (HD) with ED50 values near 1 μM. AT8 and pThr217 are commercially available antibodies for detection of hyperphosphorylated tau at specificepitopes. These tau phospho-epitopes have been associated with AD-type hyperphosphorylated tau. AT8 recognizes phosphorylated paired helical filament tau and recognizes tau phosphorylated at both serine 202 and threonine 205. No cross-reactivity with unphosphorylated tau is observed. pThr217 recognizes phosphorylated tau at threonine 217.ResultsNeuronal production of nitric oxide radicals can be prevented by NOS inhibitionTo explore the effect of NOS inhibition in hippocampal neurons, an assay to monitor NO production in neurons was established using the fluorescent dye diaminofluorescein-FM diacetate (DAF-FM DA)70,71. L-Glutamate and D-serine, co-agonists of the NMDAR, were administered to neurons as a positive control for nNOS activation72. An increased trend in the integrated fluorescent intensity was observed in the treated condition as compared to the control, indicating glutamate-induced NO production (FIG. 4, 540x, ns).The general NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was used as a negative control for NO production. L-NAME pretreatment showed a trend toward reduction of integrated fluorescence intensity, reflecting the suppression of NO production by NOS inhibition (FIG. 5 A, 100%, ns).An isoform of NOS found in immune cells, iNOS, can produce large amounts of NO50. Since this signal could significantly mask nNOS activation, the inventors verified that most NO production stimulated by glutamate was occurring in neurons, and not in nearby glial cells. The inventors detected NO using DAF-FM DA, then fixed and labeled cells with an antibody against P-III tubulin to selectively stain neurons. Results indicated that most glutamate- stimulated NO production occurs in neurons (FIG. 6, median 66%). Finally, higher- magnification images were obtained (FIG. 5B). The blue fluorescent 4',6-diamidino-2- phenylindole (DAPI) stain was added to mark nuclei. NO production was detected in numerous 3-10 pm ovular regions close to the cell body in many cells, and with diffuse cell-body staining in some cells. These observations are consistent with the description of nNOS subcellular distribution by Roth et al., who observed brain nNOS localized in some cases to mitochondrial membranes, or in a more diffuse cellular distribution73.Cellular expression of nNOS was variable across cell cultureAn antibody for nNOS (3G6B10) was used to measure cell-to-cell trends in expression of nNOS. A 5-fold variation was observed in nNOS expression across different cells (FIG. 7). This suggests that some cells may be more vulnerable to any nNOS-mediated neurodegeneration.Inhibition of nNOS blocked AβO-induced tau phosphorylation at Thr205 and pSer396 Therefore, to determine whether nNOS activation was required to mediate AβO- induced tau phosphorylation, mature neurons were treated with vehicle or 500 nM AβO, with or without 100 µM nNOS inhibitor (HD-3-86, 30 min pretreatment) and probed for AβO binding (NU4) and tau phosphorylation at Thr205. Although it did not alter AβO binding (FIG. 8a, c, ns), nNOS inhibition significantly prevented downstream tau phosphorylation at Thr205 (FIG.8b, 108% reduction, p <0.0001). In a separate experiment, in a separate neuron culture, nNOS inhibition also prevented tau phosphorylation at Ser396 (FIG. 9, 97%, p < 0.0001). Based on these results, the inventors conclude that nNOS is required for tau phosphorylation at both Thr205 and Ser396, two AD-relevant phosphorylation sites. nNOS inhibition increases synaptic puncta from baseline In AD, the degeneration of synaptic spines correlates closely with memory loss74-77. Therefore, to determine whether nNOS inhibition could alter the number of synaptic spines on neurons, phalloidin staining was used to label filamentous actin, which is highly enriched in synapses. Treatment with the nNOS inhibitor HD-3-86, even in the presence of crosslinked AβOs, induced an increase in synaptic spine density (FIG.10, intensity increased 1.5-fold, p = 0.0499). This supports the conclusion that nNOS inhibition could protect or even enhance synapses. nNOS inhibition reduces Aβ-derived AβO buildup We also tested the effect of nNOS inhibition on AβO buildup along neurons when Aβ monomer was applied. In two separate experiments using different cell cultures, nNOS inhibition reduced AβO buildup (FIG.11, 54%, p = 0.0057). This supports the conclusion that nNOS inhibition protects cells not only from AβO-induced tau phosphorylation, but also from upstream AβO buildup. Summary of results The results obtained in this study suggest that NMDAR-stimulated NO production in neurons could be prevented by NOS inhibition and that the majority of NO production was localized to cell body-adjacent regions within neurons. Additionally, cellular expression of nNOS varies across hippocampal cell culture, and AβO-induced tau phosphorylation at pThr205 and pSer396 is dependent upon nNOS activity. Finally, nNOS inhibition increased the number of synaptic spines on neurons and suppressed Aβ-derived AβO buildup. Taken together, these results identify nNOS activity as a contributor to AβO buildup in neurons and AβO-induced tau phosphorylation, and support nNOS inhibition as a potential therapeutic strategy to protect against these pathologic hallmarks of AD.Mechanisms of interestThe obtained results suggest several potential mechanisms for nNOS-dependent tau phosphorylation, synapse growth suppression, and A0 oligomerization. In these mechanisms, significant evidence indicates the involvement of NO, but the role of nNOS has not previously been established. First, two major tau kinases are activated by nitric oxide: cyclin-dependent kinase 5 and glycogen synthase kinase-3p. Once tau is phosphorylated, Fyn kinase likely contributes to enhancement of nNOS-dependent tau phosphorylation. In addition to phosphorylation, we hypothesize a potential role for nNOS in triggering tau acetylation by GAPDH nitrosylation. S-nitrosylation of Dexrasl could also be involved in propagating effects of nNOS activation, including tau phosphorylation. To suppress synapse growth, we hypothesize that nNOS may be involved in suppressing mTOR activity downstream of the NMD AR. Finally, nNOS may contribute to AP oligomerization by triggering release of zinc, or by inhibition of the chaperone protein disulfide isomerase. These proposed mechanisms lay a foundation for potential future experiments to identify the role of downstream effectors in nNOS-dependent, AD-related processes. nNOS-dependence of tau phosphorylation at Thr205 - a potential role for Cdk5Our results demonstrate that ApO-induced tau phosphorylation at Thr205 is nNOS- dependent. We hypothesize that S-nitrosylation of cyclin-dependent kinase 5 (Cdk5), a tau kinase, is the mechanism by which nNOS mediates ApO-induced tau phosphory lation at Thr205 (FIG. 12). The tau phosphorylation site at Thr205 comprises a major phosphorylation target of Cdk5. S-nitrosylation of Cdk5, which is elevated in AD63, enhances the activity of Cdk5. Qu et al. demonstrated that application of Ap to neurons triggers S-nitrosylation of Cdk5, which could potentially be mediated by nNOS downstream of NMDA receptor activation, and which caused downstream synaptic spine loss63. Conversely, a mutation of Cdk5 that prevented nitrosylation, as well as a nitric oxide synthase inhibitor, resulted in protection against synaptic spine loss63. In AD, expression of the Cdk5 activator protein p25 is increased; p25 formation can be triggered when Ap binding at synapses causes intracellular calpain activation78,79.Town et al. also found that Ap-induced Cdk5 overactivation can cause tau hyperphosphorylation, but this effect was attributed to calpain-mediated increases in p2563-700’83. Given these results, nNOS-dependent S-nitrosylation of Cdk5 may be a major contributor to ApO-induced tau phosphory lation. Future studies to identify yvhether nNOS inhibition diminishes ApO-induced Cdk5 S-nitrosylation, and whether nNOS inhibitors are still able to prevent ApO-induced tau phosphorylation at Thr205 in the presence of constitutively active Cdk5, could further support this possible explanation.nNOS-dependence of tau phosphorylation at Ser396 - a potential role for GSK-3POur experiments also show that ApO-induced tau phosphorylation at Ser396 is nNOS- dependent. This result implicates nNOS-dependent activation of a second tau kinase, glycogen synthase kinase-3p (GSK-3P, FIG. 12)62,84. Ser396 is a major phosphorylation target of GSK- 3p. Zhang et al. determined that application of NO to neurons resulted in GSK-3p-dependent tau phosphorylation at Ser396 / 40474. Additionally, the distribution of active GSK-3P in the AD brain matches patterns of tau phosphorylation85. Further, inhibition of GSK-3P and knockdow n of GSK-3P expression both protected neurons against AP-induced tau phosphorylation86’87. Future studies to determine whether nNOS inhibitors are still able to prevent ApO-induced tau phosphorylation at Ser396 in the presence of constitutively active GSK-3P would establish whether nNOS-dependent tau phosphorylation at this site relies on activation of GSK-3P. Potential Fyn kinase contribution to nNOS signalingAs previously discussed, the inventors observed that nNOS is necessary' for ApO- induced tau phosphorylation at two key AD-relevant sites. Tau hyperphosphorylation causes tau to detach from microtubules and redistribute from the axon to the cell soma and dendrites88-90 26,27,91-94 Therejautethers Fyn to the PSD-95 scaffold protein, which facilitates phosphorylation oftheNMDAR by Fyn, enhancing PSD-95 / NMDAR stability (FIG. 13). Ittner et al. found that Fyn was necessary' for AP-induced neurotoxicity94-96. Given that nNOS is physically and functionally linked to the NMD AR by PSD-95, this enhanced association likely contributes to the observed nNOS-dependent AD-relevant processes the inventors observed. Future experiments to determine whether Fyn is necessary for tau phosphorylation at both Ser396 and Thr205, and whether nNOS inhibition prevents Fyn-mediated NMDAR phosphorylation would help to establish the interaction of Fyn with this newly established role for nNOS.Potential role of nNOS in tau acetylationRecently, in addition to phosphorylation, acetylation of tau has been identified as a post-translational modification which is elevated in AD brains97. Acetylation impairs tau function and contributes to aggregation and toxicity mediated by tau98-102. Sen and coworkers showed that, in cortical neurons. Ap stimulates nitrosylation of GAPDH, a modification which is also enhanced in the brains of AD patients103. Nitrosylated GAPDH activates the tau acetylase p300 and concurrently inhibits the tau deacetylase SIRT1; as a result, tau acetylation is enhanced via a nitrosylation-dependent pathway (FIG. 14). Conversely, use of the GAPDH nitrosylation inhibitor omigapil prevented AP-induced tau acetylation and memory impairmentin mice. These observations suggest that nNOS could also contribute to pathogenic tau acetylation.Dexrasl activation and tau nitration as potential effects of nNOSAnother adaptor protein, the C-terminal PDZ ligand of nitric oxide synthase (CAP0N / N0S1AP) also acts as a scaffold to promote interaction of nNOS with additional targets (FIG. 15)104. Binding of nNOS to CAPON is triggered by NMD AR activation and has been shown to help potentiate NMD AR signaling105. CAPON expression is upregulated in CAI pyramidal cells of the AD brain, and CAPON deficiency ameliorated AD-related phenotypes in an AD model mouse65 106. One ligand coupled to nNOS by CAPON is Dexrasl. Zhang et al. demonstrated that A0 species enhance the nNOS-CAPON interaction, leading to nitrosylation of Dexrasl64. Dexrasl S-nitrosylation triggers damage to mitochondria, causing AP-induced neurodegeneration in their model. Further, Hashimoto et al. reported that tau binds to CAPON, and that nNOS-CAPON interaction allows increased nitration of tau at Tyr29, promoting tau phosphorylation65. nNOS increases the number of synapsesThe inventors also observed that nNOS inhibition stimulates an increase in synaptic spines. Li et al. demonstrated that the mechanism for this synaptic spine formation involves activation of the mTOR pathway107. However, adding complexity to this regulatory pathway, Ruddy et al. reported that, while acute NMD AR inhibition increases spine density, long-term administration at low levels reduces spine density110. This may explain the reduction in spine density7that is observed in NMD AR knockout animals111. nNOS inhibition can prevent ApO buildup from monomerOur results demonstrating that nNOS inhibition can reduce dendritic Apo buildup from monomer in cell culture match observations by Deshpande et al. showing that synaptic formation of ApOs is activity-dependent, and that targeting to synapses can be prevented by NMD AR inhibitors112. They implicated release of zinc as a potential mediator of this ApO formation, given that zinc can stabilize certain ApO species67. Bossy-Wetzel et al. showed that NO can stimulate release of free zinc to the cytoplasm68. Future experiments to determine whether nNOS inhibitors alter zinc release in neurons, and whether addition of excess zinc could prevent the protective effect of nNOS inhibition would establish whether nNOS acts by preventing zinc-dependent ApO formation. Alternatively, S-nitrosylation of protein disulfide isomerase (PDI). a chaperone which prevents protein aggregation, has also been observed in AD66.Therapeutic applications of nNOS inhibitorsIn conclusion, this work establishes a role for the activation of nNOS in ApO-induced tau phosphorylation, APO formation, and modulation of spine morphology. Although alterations of calcium homeostasis induced by ApOs have previously been reported113, and nitrosylation and nitration are implicated in AD pathology62'68, a direct role for nNOS in this mechanism has not been previously established. This work suggests that nNOS inhibition could provide a strategy to block ApO-related neurodegeneration. Important considerations for therapeutic application of nNOS inhibitors will be the effect of long-term administration, and the identification of an appropriate therapeutic window in which normal function is preserved, but aberrant signaling is prevented. These results demonstrate that multiple forms of AD neurodegeneration are nNOS-dependent and can be disrupted by specific inhibition of nNOS. MethodsNeuron culture in phenol red-free media for NO assays1 pair of combined El 8 Sprague-Dawley rat hippocampus, cortex, and sub-ventricular zone tissue was obtained fresh from Transnetyx (SDEHCV) and cultured according to the manufacturer’s protocol, based on the procedures from Brewer et al.114,115. Cells were diluted to 0.2-0.4 million / mL and plated at 100 pL / well directly onto sterile pre-coated 96-well plates (Fisher Scientific 07-000-190). This yielded a density of approximately 20,000-40,000 cells / well. Cells were fed every seven days by addition of 35-50 pL phenol red-free NbActiv4 (Transnetyx NB4 and NB4PR500).DAF-FM DA assay for NO production in neuronsNitric oxide (NO) production in neurons was measured using the fluorescent dye diaminofluorescein-FM diacetate (DAF-FM DA, Thermo Fisher Scientific D23844) according to the previous protocols by Nott and Kolarow70’71. DAF-FM DA was added to cells in transparent conditioned media (80 μM, 37 °C, 2h, dark). Cells were subsequently washed twice with Hanks’ Balanced Salt Solution. In some experiments, the general NOS inhibitor NG-nitro- L-arginine methyl ester (L-NAME) or vehicle was applied (200 μM, 30 min, 37 °C, dark). Then 100 μM L-glutamate and 10 μM D-serine were applied in transparent conditioned media (5 h, 37 °C)72. Live cells were imaged using the ImageXPress microscope in the Northwestern High- Throughput Analysis Laboratory at lOx objective. The total integrated fluorescence intensity for each well was quantified using ImageJ.To co-stain for neurons, the cells were next fixed by addition of 3.7% formaldehyde 1: 1, 10 min, and subsequent replacement of this mixture with 3.7% formaldehyde for 10 min. Cells were then blocked using 10% normal goat serum in PBS with 0.1% Triton X-100 for 30-45min, RT. A primary mouse antibody against P-III tubulin (Promega G712A, 1:2000), which selectively stained neurons, was applied in blocking buffer at 4 °C overnight. After washing, this antibody was visualized using the Alexa 647 goat anti-mouse antibody (Invitrogen Al 1029, 1 :2000, 3h, RT, dark). In some cases, blue fluorescent 4',6-diamidino-2-phenylindole (DAPI) stain was added as well to mark nuclei. These were imaged using the ImageXPress microscope in the Northwestern High-Throughput Analysis Laboratory’ at lOx and 40x objectives.Neuron culture on coverslips for immunofluorescence of fixed cells1 pair of combined El 8 Sprague-Dawley rat hippocampus, cortex, and sub-ventricular zone tissue was obtained fresh from Transnetyx (SDEHCV) and cultured according to the manufacturer's protocol, based on the procedures from Brewer et al.114,115. Cells were diluted to 0.11-0.22 million / mL and plated at 0.15 mL / coverslip directly onto sterile poly-D-lysine (Sigma-Aldrich P6407) coated 12 mm circular coverslips (Fisher Scientific 12-545-80P or 12- 545-81P), which were prearranged 4 / dish in 35 mm culture dishes (Fisher Scientific 08-772A). This yielded a density of approximately 15.000-30.000 cells / cm2. Cells were fed 4 days after plating by half-media exchange with NbActivl (Transnetyx NB1) and subsequently every 2-3 days by half-media exchange with NbActivl.Cell treatment and immunofl uorescent detection of pTau levelsNeurons were pretreated at 12-21 div with 100 μM HD-3-86 or DMSO vehicle as control. ApOs. made according to the protocol of Lambert et al., or F12 media vehicle as control (Caisson Labs HFL05) were added to cells for 4-6 h, 37 °C116. For immunofluorescence, cells were fixed by addition of an equal volume of 3.7% formaldehyde solution (Sigma- Aldrich F8775-500) for 10 min, followed by replacement with 3.7% formaldehyde for 10 min. Coverslips were washed three times with PBS and blocked with 5% normal goat serum (Fisher Scientific ICN19135680), 0.1% Tnton X-100 (Sigma-Aldnch T9284-500ML) in PBS for 1 h at RT. Primary antibodies NU4 (1 :2000, Klein lab), anti-tau pThr205 (1 :500, GeneTex GTX31124), anti-tau pSer396 (1 :500, Thermo Fisher Scientific 710298), and / or anti-nNOS (1 :500 based on FIG. 16, Thermo Fisher Scientific 372800) were applied overnight in blocking buffer at 4 °C.Coverslips were then washed three times with PBS and secondary antibodies, goat antimouse Alexa Fluor 488 (1 :2000, Fisher Scientific Al 1029) and goat anti-rabbit Alexa Fluor 568 (1:2000, Thermo Fisher Scientific Al 1036) were added in a solution of 10% blocking buffer in PBS. 3 h at RT in the dark. Coverslips were washed three times and mounted in Prolong Diamond Antifade mountant with DAPI (Thermo Fisher Scientific P36862).Some inconsistencies with the tau phosphorylation assays were observed over time. The best results were often obtained using fresh antibody, and some batches of antibody failed to show ApO-induced tau phosphorylation. After initial freeze-thaw, the antibody against pSer396 (Thermo Fisher Scientific 710298) failed to show tau phosphorylation (data not shown), suggesting that aliquoting to avoid freeze-thaws is crucial for its use.For this analysis, crosslinked ApOs (xLApOs) were used due to their higher observed binding to cells. xLApOs were prepared according to the method of Cline et al., followed by 6 rounds of buffer exchange into PBS to remove excess reagents and to enrich Apt)s117The AT8 antibody (Thermo Fisher Scientific MN1020), which detects tau phosphorylation at Thr205, was initially found to detect small but significant increases in tau phosphorylation when 500 nM xLApO was administered to mature cells at 21 div for 6 h (FIG. 17 a, p = 0.0006, 1.4x). Additional tests of different aliquots, which likely experienced different amounts of freeze / thawing, showed no ApO-induced tau phosphorylation for one aliquot (FIG. 17 b, p = 0.82), while a significant increase was observed for another aliquot (FIG. 17 c, p = 0.0083, 4.4x). This result supports the conclusion that different aliquots of the same antibody might label phosphorylated tau with different efficacy, likely due to a loss of efficacy after multiple freeze-thaws. For this reason, antibodies were then routinely aliquoted to prevent high numbers of freeze-thaw cycles.Synaptic spine measurement with phalloidinNeurons at 21 div were pretreated with 100 μM HD-3-86 or DMSO vehicle as control. ApOs or Fl 2 media (Caisson Labs HFL05) vehicle as control were added to cells for 30 min at 37 °C. Cells were w ashed with PBS, and w ere then fixed by addition of an equal volume of 3.7% formaldehy de solution (Sigma-Aldrich F8775-500) for 10 min, follow ed by replacement with 3.7% formaldehyde for 10 min. Cells were washed with PBS. and then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich T9284-500ML) in PBS for 5 min at RT. Cells were washed again with PBS, and then phalloidin reconstituted at 14 μM in 100% methanol w as applied to cells at 100 nM in PBS for 30 min at RT in the dark. Coverslips w ere w ashed three times with PBS and mounted in Prolong Diamond Antifade mountant with DAPI (Thermo Fisher Scientific P36862). Slides were stored protected from light at 4 °C for less than 2 weeks pnor to imaging.ApO binding assayMature hippocampal neurons at 21-22 div were pretreated first for 30 min with 100 μM HD-3-86 or the corresponding DMSO vehicle in conditioned media. Then 500 nM Ap or DMSO vehicle was applied for 30 min. To add Ap, half of the media was removed from eachdish, and a 2x solution of the next treatment was prepared in that conditioned media, which was then added back to the dish and mixed well. For immunofluorescence, cells were washed with sterile PBS prior and subsequently fixed by addition of an equal volume of 3.7% formaldehyde solution (Sigma- Aldrich F8775-500) for 10 min, followed by replacement with 3.7% formaldehyde for 10 min. Coverslips were washed three times with PBS and blocked with 5% normal goat serum (Fisher Scientific ICN19135680), 0.1% Triton X-100 (Sigma- Aldrich T9284-500ML) in PBS for 1 h at RT or 4 °C overnight. Primary antibodies NU2 (1 :2000, Klein lab) and anti-MAP2 (1 : 10,000, EnCor Biotechnology CPCA-MAP2) were applied overnight in blocking buffer at 4 °C. Coverslips w ere then washed three times with PBS and secondary antibodies, goat anti-mouse Alexa Fluor 488 (1:2000, Fisher Scientific Al 1029) and goat anti-chicken Alexa Fluor 568 (1:2000, Thermo Fisher Scientific Al 1041) were added in a solution of 10% blocking buffer in PBS for 3 h at RT in the dark. Coverslips were washed three times and mounted in Prolong Diamond Antifade mountant with DAPI (Thermo Fisher Scientific P36862).Quantification was conducted on immunofluorescent images in which a structural reference antibody, most commonly MAP2, but also including P-3 tubulin, was used as an indicator of neuronal processes. ImageJ was used for analysis, using macros included in the Appendix. Briefly, tiff images w ere assigned random numbers to avoid bias during analysis, and then cell bodies were manually traced and deleted for each image to eliminate signal from nonspecific antibody accumulation in the cell body. Then, the signal from different wavelengths was split. To calculate total dendrite length in each image, a Gaussian blur was applied to the MAP2 signal, and the signal above an equal threshold for each image was skeletonized, and the total length of the skeleton w as measured. To calculate the total Apo puncta along processes, again a Gaussian blur was applied to the dendrite signal, and the signal above an equal threshold was selected. Then, the signal was dilated to encompass the area around the dendrites by 15 dilations. The area around the dendrites was used as a selection, and within the selection area, ImageJ particle analysis and summary7were used to count the NU2 puncta above an equal threshold for each image. Thresholds were chosen based on one or two positive and negative control images. 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The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency betw een a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMS We claim:

1. A method for reducing or preventing AβO-induced tau phosphorylation comprising contacting neurons with an effective amount of a neuronal nitric oxide synthase inhibitor.

2. The method of claim 1, wherein the method is for reducing or preventing AβO-induced tau phosphorylation at Thr205.

3. The method of claim 1, wherein the method is for reducing or preventing AβO-induced tau phosphorylation at Ser396.

4. The method of any one of claims 1-3, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in vitro or ex vivo.

5. The method of claims 1-3, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder, wherein the neurodegenerative disease or disorder is optionally Alzheimer’s Disease.

6. The method of claim 5, wherein the subject is in need of a treatment for reducing or preventing AβO-induced tau phosphorylation.

7. The method of any one of claims 1-6, wherein the neuronal nitric oxide synthase inhibitor is a compound of a Formula (I) or a salt or solvate thereof:1-C6-alkyl, C1-C6-alkoxy, halogen, or haloalkyl, represents a single, double, or triple bond;Y is substituted aryl or substituted heteroaryl, wherein Y is substituted at one or more ring positions with halogen or a substituent having a formula —Z—Ra; or Y has a formula — Z—Ra; Z is selected from C1-C6-alkyl, C2-C6-alkenyl, and C2-C6-alkynyl; Rais selected from amino, alkylamino, dialkylamino, or a 4-6 membered heterocycle which contains at least one nitrogen atom and which heterocycle is optionally substituted at one or more positions with alkyl, alkoxy, or halogen.

8. The method of claim 7, wherein the neuronal nitric oxide synthase inhibitor is HD-3-86, .

9. A method for increasing synaptic spine density comprising contacting neurons with an effective amount of a neuronal nitric oxide synthase inhibitor in the presence of AβO.

10. The method of claim 9, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in vitro or ex vivo.

11. The method of claim 9, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder, wherein the neurodegenerative disease or disorder is optionally Alzheimer’s Disease.

12. The method of claim 11, wherein the subject is in need of a treatment for increasing synaptic spine density.

13. The method of any one of claims 9-12, wherein the neuronal nitric oxide synthase inhibitor is a compound of a Formula (I) or a salt or solvate thereof:l, C1-C6-alkoxy, halogen, or haloalkyl,represents a single, double, or triple bond; Y is substituted aryl or substituted heteroaryl, wherein Y is substituted at one or more ring positions with halogen or a substituent having a formula —Z—Ra; or Y has a formula — Z—Ra; Z is selected from C1-C6-alkyl, C2-C6-alkenyl, and C2-C6-alkynyl; Rais selected from amino, alkylamino, dialkylamino, or a 4-6 membered heterocycle which contains at least one nitrogen atom and which heterocycle is optionally substituted at one or more positions with alkyl, alkoxy, or halogen.

14. The method of claim 13, wherein the neuronal nitric oxide synthase inhibitor is HD-3-86, .

15. A method for reducing or preventing AβO buildup comprising contacting neurons with an effective amount of a neuronal nitric oxide synthase inhibitor in the presence of Aβ monomer.

16. The method of claim 14, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in vitro or ex vivo.

17. The method of claim 14, wherein the neurons are contacted with the neuronal nitric oxide synthase inhibitor in a subject in need of a treatment for a neurodegenerative disease or disorder, wherein the neurodegenerative disease or disorder is optionally Alzheimer’s Disease.

18. The method of claim 17, wherein the subject is in need of a treatment for increasing synaptic spine density.

19. The method of any one of claims 15-18, wherein the neuronal nitric oxide synthase inhibitor is a compound of the Formula (I) or a salt or solvate thereof: n, C1-C6-alkyl, C1-C6-alkoxy, halogen, or haloalkyl,represents a single, double, or triple bond; Y is substituted aryl or substituted heteroaryl, wherein Y is substituted at one or more ring positions with halogen or a substituent having a formula —Z—Ra; or Y has a formula — Z—Ra; Z is selected from C1-C6-alkyl, C2-C6-alkenyl, and C2-C6-alkynyl; Rais selected from amino, alkylamino, dialkylamino, or a 4-6 membered heterocycle which contains at least one nitrogen atom and which heterocycle is optionally substituted at one or more positions with alkyl, alkoxy, or halogen.

20. The method of claim 19, wherein the neuronal nitric oxide synthase inhibitor is HD-3-86, .. the preceding claims wherein the neuronal nitric oxide synthase inhibitor is of a formula:selected from Cl, F, OMe, CH2F, CHF2, and CF3.

22. The method of any of the preceding claims wherein the neuronal nitric oxide synthase inhibitor is of a formula:

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