Anti-Neurodegenerative Benefits of Acetylcholinesterase Inhibitors in Alzheimer's Disease: Nexus of Cholinergic and Nerve Growth Factor Dysfunction.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is increasingly viewed as a complex multi-dimensional disease without effective treatments. Recent randomized, placebo-controlled studies have shown volume losses of ~0.7% and ~3.5% per year, respectively, in the basal cholinergic forebrain (CBF) and hippocampus in untreated suspected prodromal AD. One year of donepezil treatment reduced these annualized rates of atrophy to about half of untreated rates. Similar positive although variable results have also been found in volumetric measurements of the cortex and whole brain in patients with mild cognitive impairment as well as more advanced AD stages after treatments with all three currently available acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, and galantamine). Here we review the anti-neurodegenerative benefits of AChE inhibitors and the expected parallel disease-accelerating impairments caused by anticholinergics, within a framework of the cholinergic hypothesis of AD and AD-associated loss of nerve growth factor (NGF). Consistent with the "loss of trophic factor hypothesis of AD," we propose that AChE inhibitors enhance acetylcholine-dependent release and uptake of NGF, thereby sustaining cholinergic neuronal viability and thus slowing AD-associated degeneration of the CBF, to ultimately delay dementia progression. We propose that improved cholinergic therapies for AD started early in asymptomatic persons, especially those with risk factors, will delay the onset, progression, or emergence of dementia. The currently available competitive and pseudo- irreversible AChE inhibitors are not CNS-selective and thus induce gastrointestinal toxicity that limits cortical AChE inhibition to ~30% (ranges from 19% to 41%) as measured by in vivo PET studies in patients undergoing therapy. These levels of inhibition are marginal relative to what is required for effective symptomatic treatment of dementia or slowing AD-associated neurodegeneration. In contrast, because of the inherently slow de novo synthesis of AChE in the CNS (about one-- tenth the rate of synthesis in peripheral tissues), irreversible AChE inhibitors produce significantly higher levels of inhibition in the CNS than in peripheral tissues. For example, methanesulfonyl fluoride, an irreversible inhibitor reduces CNS AChE activity by ~68% in patients undergoing therapy and ~80% in cortical biopsies of non-human primates. The full therapeutic benefits of AChE inhibitors, whether for symptomatic treatment of dementia or disease-slowing, thus would benefit by producing high levels of CNS inhibition. One way to obtain such higher levels of CNS AChE inhibition would be by using irreversible inhibitors.

Sphingosine-1-phosphate receptor-independent lung endothelial cell barrier disruption induced by FTY720 regioisomers.

RATIONALE: Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. OBJECTIVE: To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. METHODS: Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. RESULTS: FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. CONCLUSIONS: Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

A Pilot Microbiota Study in Parkinson's Disease Patients versus Control Subjects, and Effects of FTY720 and FTY720-Mitoxy Therapies in Parkinsonian and Multiple System Atrophy Mouse Models.

BACKGROUND: Parkinson's disease (PD) and multiple system atrophy (MSA) patients often suffer from gastrointestinal (GI) dysfunction and GI dysbiosis (microbial imbalance). GI dysfunction also occurs in mouse models of PD and MSA. OBJECTIVES: To assess gut dysfunction and dysbiosis in PD subjects as compared to controls, identify potential shared microbial taxa in humans and mouse models of PD and MSA, and to assess the effects of potential therapies on mouse GI microbiota. METHODS: In this human pilot study, GI function was assessed by fecal consistency/frequency measured using the Bristol Stool Form Scale and GI transit time assessed using Sitzmarks pills and abdominal radiology. Human and mouse microbiota were analyzed by extracting fecal genomic DNA followed by 16S rRNA sequencing. RESULTS: In our PD patients genera Akkermansia significantly increased while a trend toward increased Bifidobacterium and decreased Prevotella was observed. Families Bacteroidaceae and Lachnospiraceae and genera Prevotella and Bacteroides were detected in both humans and PD mice, suggesting potential shared biomarkers. In mice treated with the approved multiple sclerosis drug, FTY720, or with our FTY720-Mitoxy-derivative, we saw that FTY720 had little effect while FTY720-Mitoxy increased beneficial Ruminococcus and decreased Rickenellaceae family. CONCLUSION: Akkermansia and Prevotellaceae data reported by others were replicated in our human pilot study suggesting the use of those taxa as potential biomarkers for PD diagnosis. The effect of FTY720-Mitoxy on taxa Rikenellaceae and Ruminococcus and the relevance of S24-7 await further evaluation. It also remains to be determined if mouse microbiota have predictive power for human subjects.

FTY720-Mitoxy reduces synucleinopathy and neuroinflammation, restores behavior and mitochondria function, and increases GDNF expression in Multiple System Atrophy mouse models.

Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1 mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5-11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starch­iodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.

FTY720-Mitoxy reduces toxicity associated with MSA-like α-synuclein and oxidative stress by increasing trophic factor expression and myelin protein in OLN-93 oligodendroglia cell cultures.

Multiple system atrophy (MSA) is a fatal demyelinating disorder lacking any disease-modifying therapies. MSA pathology stems from aggregated α-synuclein (aSyn) accumulation in glial cytosolic inclusions of oligodendroglial cell (OLGs), the myelinating cells of brain. In MSA brains and in MSA animal models with aSyn accumulation in OLGs, aberrant expression of brain-derived neurotrophic factor (BDNF) and glial-cell-line-derived neurotrophic factor (GDNF) occur. Nerve growth factor (NGF) expression can also be altered in neurodegenerative diseases. It is unclear if oxidative stress impacts the viability of aSyn-accumulating OLG cells. Here, we show that OLN-93 cells stably expressing human wild type aSyn or the MSA-associated-aSyn-mutants G51D or A53E, are more vulnerable to oxidative stress. In dose response studies we found that OLN-93 cells treated 48 h with 160 nM FTY720 or our new non-immunosuppressive FTY720-C2 or FTY720-Mitoxy derivatives sustained normal viability. Also, FTY720, FTY720-C2, and FTY720-Mitoxy all stimulated NGF expression at 24 h. However only FTY720-Mitoxy also increased BDNF and GDNF mRNA at 24 h, an effect paralleled by increases in histone 3 acetylation and ERK1/2 phosphorylation. Myelin associated glycoprotein (MAG) levels were also increased in OLN-93 cells after 48 h treatment with FTY720-Mitoxy. FTY720, FTY720-C2, and FTY720-Mitoxy all prevented oxidative-stress-associated-cell-death of OLN-93 cells that lack any aSyn expression. However, only FTY720-Mitoxy protected MSA-like aSyn-expressing-OLN-93-cells against oxidative-cell-death. These data identify potent protective effects for FTY720-Mitoxy with regard to trophic factors as well as MAG expression by OLG cells. Testing of FTY720-Mitoxy in mice is thus a judicious next step for neuropharmacological preclinical development.

FTY720 Improves Behavior, Increases Brain Derived Neurotrophic Factor Levels and Reduces α-Synuclein Pathology in Parkinsonian GM2+/- Mice.

Parkinson's disease (PD) is a progressive aging disorder that affects millions worldwide, thus, disease-modifying-therapies are urgently needed. PD pathology includes α-synuclein (aSyn) accumulation as synucleinopathy. Loss of GM1 gangliosides occurs in PD brain, which is modeled in GM2 synthase transgenic mice. GM2+/- mice have low, not absent GM1 and develop age-onset motor deficits, making them an excellent PD drug testing model. FTY720 (fingolimod) reduces synucleinopathy in A53T aSyn mice and motor dysfunction in 6-OHDA and rotenone PD models, but no one has tested FTY720 in mice that develop age-onset PD-like motor problems. We confirmed that GM2+/-mice had equivalent rotarod, hindlimb reflexes, and adhesive removal functions at 9 mo. From 11 mo, GM2+/- mice received oral FTY720 or vehicle 3x/week to 16 mo. As bladder problems occur in PD, we also assessed GM2+/- bladder function. This allowed us to demonstrate improved motor and bladder function in GM2+/- mice treated with FTY720. By immunoblot, FTY720 reduced levels of proNGF, a biomarker of bladder dysfunction. In humans with PD, arm swing becomes abnormal, and brachial plexus modulates arm swing. Ultrastructure of brachial plexus in wild type and GM2 transgenic mice confirmed abnormal myelination and axons in GM2 transgenics. FTY720 treated GM2+/- brachial plexus sustained myelin associated protein levels and reduced aggregated aSyn and PSer129 aSyn levels. FTY720 increases brain derived neurotrophic factor (BDNF) and we noted increased BDNF in GM2+/- brachial plexus and cerebellum, which contribute to rotarod performance. These findings provide further support for testing low dose FTY720 in patients with PD.

Parkinsonian GM2 synthase knockout mice lacking mature gangliosides develop urinary dysfunction and neurogenic bladder.

Parkinson's disease is a neurodegenerative disorder that reduces a patients' quality of life by the relentless progression of motor and non-motor symptoms. Among the non-motor symptoms is a condition called neurogenic bladder that is associated with detrusor muscle underactivity or overactivity occurring from neurologic damage. In Parkinson's disease, Lewy-body-like protein aggregation inside neurons typically contributes to pathology. This is associated with dopaminergic neuron loss in substantia nigra pars compacta (SNc) and in ventral tegmental area (VTA), both of which play a role in micturition. GM1 gangliosides are mature glycosphingolipids that enhance normal myelination and are reduced in Parkinson's brain. To explore the role of mature gangliosides in vivo, we obtained GM2 Synthase knockout (KO) mice, which develop parkinsonian pathology including a loss of SNc dopaminergic neurons, which we reconfirmed. However, bladder function and innervation have never been assessed in this model. We compared GM2 Synthase KO and wild type (WT) littermates' urination patterns from 9 to 19 months of age by counting small and large void spots produced during 1 h tests. Because male and female mice had different patterns, we evaluated data by sex and genotype. Small void spots were significantly increased in 12-16 month GM2 Synthase KO females, consistent with overactive bladder. Similarly, at 9-12 month GM2 KO males tended to have more small void spots than WT males. As GM2 Synthase KO mice aged, both females and males had fewer small and large void spots, consistent with detrusor muscle underactivity. Ultrasounds confirmed bladder enlargement in GM2 Synthase KO mice compared to WT mice. Tyrosine hydroxylase (TH) immunohistochemistry revealed significant dopaminergic loss in GM2 Synthase KO VTA and SNc, and a trend toward TH loss in the GM2 KO periaqueductal gray (PAG) micturition centers. Levels of the nerve growth factor precursor, proNGF, were significantly increased in GM2 Synthase KO bladders and transmission electron micrographs showed atypical myelination of pelvic ganglion innervation in GM2 Synthase KO bladders. Cumulatively, our findings provide the first evidence that mature ganglioside loss affects micturition center TH neurons as well as proNGF dysregulation and abnormal innervation of the bladder. Thus, identifying therapies that will counteract these effects should be beneficial for those suffering from Parkinson's disease and related disorders.

Up-regulation of protective neuronal MicroRNAs by FTY720 and novel FTY720-derivatives.

In searching for Parkinson's disease (PD) pharmacotherapies we began studying FTY720, a food and drug administration (FDA) approved drug. We also created derivatives, FTY720-C2 and FTY720-Mitoxy, and began assessing them. Here we treated dopaminergic MN9D cells with FTY720s then measured microRNA (miRNA) levels by PCR arrays. We discovered that all three FTY720s increased miR376b-3p, while FTY720-C2 also increased miR-128-3p, miR-146b-5p, miR-7a-5p, and miR-9-5p, and FTY720-Mitoxy also increased miR-30d-5p. Investigations revealed that some miRNAs downregulate alpha-synuclein, while others reduce apoptosis, suggesting that FTY720s may act to reduce synucleinopathy and dopaminergic neuron loss in PD and related disorders.

Could α-Synuclein Modulation of Insulin and Dopamine Identify a Novel Link Between Parkinson's Disease and Diabetes as Well as Potential Therapies?

Characterizing the normal function(s) of the protein α-Synuclein (aSyn) has the potential to illuminate links between Parkinson's disease (PD) and diabetes and also point the way toward new therapies for these disorders. Here we provide a perspective for consideration based on our discovery that aSyn normally acts to inhibit insulin secretion from pancreatic ß-cells by interacting with the Kir6.2 subunit of the ATP-sensitive potassium channel (K-ATP). It is also known that K-ATP channels act to inhibit brain dopamine secretion, and we have also shown that aSyn is a normal inhibitor of dopamine synthesis. The finding, that aSyn modulates Kir6.2 and other proteins involved in dopamine and insulin secretion, suggests that aSyn interacting proteins may be negatively impacted when aSyn aggregates inside cells, whether in brain or pancreas. Furthermore, identifying therapies for PD that can counteract dysfunction found in diabetes, would be highly beneficial. One such compound may be the multiple sclerosis drug, FTY720, which like aSyn can stimulate the activity of the catalytic subunit of protein phosphatase 2A (PP2Ac) as well as insulin secretion. In aging aSyn transgenic mice given long term oral FTY720, the mice had reduced aSyn pathology and increased levels of the protective molecule, brain derived neurotrophic factor (BDNF) (Vidal-Martinez et al., 2016). In collaboration with medicinal chemists, we made two non-immunosuppressive FTY720s that also enhance PP2Ac activity, and BDNF expression (Vargas-Medrano et al., 2014; Enoru et al., 2016; Segura-Ulate et al., 2017a). FTY720 and our novel FTY720-based-derivatives, may thus have therapeutic potential for both diabetes and PD.

Recombinant α- ß- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays.

α-Synuclein (aSyn), ß-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo.

FTY720-derivatives do not induce FTY720-like lymphopenia.

FTY720 is an immunosuppressive multiple sclerosis (MS) drug that stimulates the expression of neuroprotective brain-derived-neurotrophic-factor (BDNF). In vivo preclinical data suggest that FTY720 could be beneficial for treating Parkinson's patients, though its immunosuppressive effects might limit its efficacy. Two novel FTY720-derivatives, FTY720-C2 and FTY720-Mitoxy, also stimulate BDNF expression and enter brain like FTY720 but are not phosphorylated, suggesting they will not produce FTY720-like immunosuppression. Using FTY720 as a positive control, we measured low and high dose FTY720-derivatives, which did not stimulate FTY720-like lymphopenia or immunosuppressive signaling. These findings support the further preclinical assessment of the derivatives as potential novel Parkinson's therapies.

FTY720 (Fingolimod) reverses α-synuclein-induced downregulation of brain-derived neurotrophic factor mRNA in OLN-93 oligodendroglial cells.

Multiple system atrophy (MSA) is a demyelinating neurodegenerative disorder characterized by accumulation of aggregated α-synuclein (aSyn) inside oligodendrocyte precursors, mature oligodendroglia, and neurons. MSA dysfunction is associated with loss of trophic factor production by glial and neuronal cells. Here, we report that recombinant wild type human aSyn uptake by OLN-93, an oligodendroglia cell-line, reduced brain-derived neurotrophic factor (BDNF) expression. Furthermore, OLN-93 cells stably transfected with human wild type or an MSA-associated mutant aSyn, A53E that produces neuronal and glial inclusions, reduced BDNF mRNA to nearly unmeasurable qPCR levels. Curiously, another MSA-associated aSyn mutant, G51D that also produces neuronal and glial inclusions, caused only a trend toward BDNF mRNA reduction in transfected OLN-93 cells. This suggests that oligodendrocyte-associated BDNF loss occurs in response to specific aSyn types. Treating OLN-93 cells with 160 nM FTY720 (Fingolimod, Gilenya®), a Food and Drug Administration (FDA) approved therapeutic for multiple sclerosis, counteracted BDNF downregulation in all aSyn OLN-93 cells. FTY720 also restored BDNF mRNA in OLN-93 cells treated with recombinant aSyn, as measured by qPCR or semiquantitatively on agarose gels. Immunoblots confirmed that FTY720 increased histone 3 acetylation in OLN-93, and chromatin immunoprecipitation assays showed increased acetylated histone 3 at BDNF promoter 1 after FTY720. Moreover, OLN-93 cells treated with valproic acid, a classic histone deacetylase inhibitor, confirmed that increasing acetylated histone 3 levels increases BDNF expression. Cumulatively, the data suggest that FTY720-associated histone deacetylase inhibition stimulates BDNF expression in oligodendroglial cells, raising the possibility that MSA patients may also benefit by treatment with FTY720.

Cholinesterase Inhibitor Therapy in Alzheimer's Disease: The Limits and Tolerability of Irreversible CNS-Selective Acetylcholinesterase Inhibition in Primates.

Irreversible acetylcholinesterase (AChE) inhibition accumulates to high levels in the central nervous system (CNS) because AChE turnover in the brain is much slower than in peripheral tissues. As expected from this CNS selectivity, the irreversible AChE inhibitor methanesulfonyl fluoride (MSF) produces significant cognitive improvement in Alzheimer's disease patients without the gastrointestinal toxicity that plagues other AChE inhibitors. However, without dose-limiting gastrointestinal toxicity, one shortcoming of the prior human studies of MSF is that the upper limits of CNS AChE inhibition that might be tolerated could not be tested. Therefore, in this study, monkeys were treated with escalating intramuscular (IM) doses of MSF that culminated with several weeks of 1.5 mg/kg dosing, more than eight times the prior human clinical dose, still without signs of toxicity. Brain biopsies showed that ∼80% AChE inhibition had been produced and that the new synthesis of cortical AChE had a half-time (t1/2) of ∼12 days. A single IM dose of 1.5 mg/kg MSF produced ∼59% inhibition in cerebrospinal fluid (CSF) AChE as measured one day later. This corresponds to a peak of ∼80% inhibition in CSF AChE at the time of the injection, recovering with a t1/2 of 2.4 days. Computational analyses suggest that MSF at clinically relevant doses could theoretically produce a steady-state AChE inhibition between 65% and 85% in the CNS. These data suggest that the full therapeutic advantage of AChE inhibition therapy can be realized without interference from dose-limiting gastrointestinal toxicity if an irreversible inhibitor is employed.

FTY720 Attenuates 6-OHDA-Associated Dopaminergic Degeneration in Cellular and Mouse Parkinsonian Models.

FTY720 (fingolimod) is the first oral drug approved for treating relapsing-remitting forms of multiple sclerosis. It is also protective in other neurological models including ischemia, Alzheimer's disease, Huntington disease and Rett syndrome. However, whether it might protect in a 6-hydroxydopamine (6-OHDA) mouse model associated with the dopaminergic pathology of Parkinson's disease (PD), has not been explored. Therefore, in the present study, we investigated the effects of FTY720 on 6-OHDA-induced neurotoxicity in cell cultures and mice. Here we show that FTY720 protected against 6-OHDA cytotoxicity and apoptosis in SH-SY5Y cells. We also show that prior administration of FTY720 to 6-OHDA lesioned mice ameliorated both motor deficits and nigral dopaminergic neurotoxicity, while also reducing 6-OHDA-associated inflammation. The protective effects of FTY720 were associated with activation of AKT and ERK1/2 pro-survival pathways and an increase in brain derived neurotrophic factor (BDNF) expression in vitro and in vivo. These findings suggest that FTY720 holds promise as a PD therapeutic acting, at least in part, through AKT/ERK1/2/P-CREB-associated BDNF expression.

Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy.

Parkinson's disease (PD) is a neurodegenerative aging disorder in which postmortem PD brain exhibits neuroinflammation, as well as synucleinopathy-associated protein phosphatase 2A (PP2A) enzymatic activity loss. Based on our translational research, we began evaluating the PD-repurposing-potential of an anti-inflammatory, neuroprotective, and PP2A stimulatory oral drug that is FDA-approved for multiple sclerosis, FTY720 (fingolimod, Gilenya®). We also designed two new FTY720 analogues, FTY720-C2 and FTY720-Mitoxy, with modifications that affect drug potency and mitochondrial localization, respectively. Herein, we describe the metabolic stability and metabolic profiling of FTY720-C2 and FTY720-Mitoxy in liver microsomes and hepatocytes. Using mouse, rat, dog, monkey, and human liver microsomes the intrinsic clearance of FTY720-C2 was 22.5, 79.5, 6.0, 20.2 and 18.3 µL/min/mg; and for FTY720-Mitoxy was 1.8, 7.8, 1.4, 135.0 and 17.5 µL/min/mg, respectively. In hepatocytes, both FTY720-C2 and FTY720-Mitoxy were metabolized from the octyl side chain, generating a series of carboxylic acids similar to the parent FTY720, but without phosphorylated metabolites. To assess absorption and distribution, we gave equivalent single intravenous (IV) or oral doses of FTY720-C2 or FTY720-Mitoxy to C57BL/6 mice, with two mice per time point evaluated. After IV delivery, both FTY720-C2 and FTY720-Mitoxy were rapidly detected in plasma and brain; and reached peak concentrations at the first sampling time points. After oral dosing, FTY720-C2 was present in plasma and brain, although FTY720-Mitoxy was not orally bioavailable. Brain-to-plasma ratio of both compounds increased time-dependently, suggesting a preferential partitioning to the brain. PP2A activity in mouse adrenal gland increased ~2-fold after FTY720-C2 or FTY720-Mitoxy, as compared to untreated controls. In summary, FTY720-C2 and FTY720-Mitoxy both (i) crossed the blood-brain-barrier; (ii) produced metabolites similar to FTY720, except without phosphorylated species that cause S1P1-mediated-immunosuppression; and (iii) stimulated in vivo PP2A activity, all of which encourage additional preclinical assessment.

FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.

Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.

The contribution of alpha synuclein to neuronal survival and function - Implications for Parkinson's disease.

The aggregation of alpha synuclein (α-syn) is a neuropathological feature that defines a spectrum of disorders collectively termed synucleinopathies, and of these, Parkinson's disease (PD) is arguably the best characterized. Aggregated α-syn is the primary component of Lewy bodies, the defining pathological feature of PD, while mutations or multiplications in the α-syn gene result in familial PD. The high correlation between α-syn burden and PD has led to the hypothesis that α-syn aggregation produces toxicity through a gain-of-function mechanism. However, α-syn has been implicated to function in a diverse range of essential cellular processes such as the regulation of neurotransmission and response to cellular stress. As such, an alternative hypothesis with equal explanatory power is that the aggregation of α-syn results in toxicity because of a toxic loss of necessary α-syn function, following sequestration of functional forms α-syn into insoluble protein aggregates. Within this review, we will provide an overview of the literature linking α-syn to PD and the knowledge gained from current α-syn-based animal models of PD. We will then interpret these data from the viewpoint of the α-syn loss-of-function hypothesis and provide a potential mechanistic model by which loss of α-syn function could result in at least some of the neurodegeneration observed in PD. By providing an alternative perspective on the etiopathogenesis of PD and synucleinopathies, this may reveal alternative avenues of research in order to identify potential novel therapeutic targets for disease modifying strategies. The correlation between α-synuclein burden and Parkinson's disease pathology has led to the hypothesis that α-synuclein aggregation produces toxicity through a gain-of-function mechanism. However, in this review, we discuss data supporting the alternative hypothesis that the aggregation of α-synuclein results in toxicity because of loss of necessary α-synuclein function at the presynaptic terminal, following sequestration of functional forms of α-synuclein into aggregates.

S-allyl cysteine activates the Nrf2-dependent antioxidant response and protects neurons against ischemic injury in vitro and in vivo.

Stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable. S-allyl cysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has been reported to possess neuroprotective effects against stroke. However, the mechanisms underlying its beneficial effects remain poorly defined. The present study tests the hypothesis that SAC attenuates ischemic neuronal injury by activating the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidant response in both in vitro and in vivo models. Our findings demonstrate that SAC treatment resulted in an increase in Nrf2 protein levels and subsequent activation of antioxidant response element pathway genes in primary cultured neurons and mice. Exposure of primary neurons to SAC provided protection against oxygen and glucose deprivation-induced oxidative insults. In wild-type (Nrf2(+/+) ) mice, systemic administration of SAC attenuated middle cerebral artery occlusion-induced ischemic damage, a protective effect not observed in Nrf2 knockout (Nrf2(-/-) ) mice. Taken together, these findings provide the first evidence that activation of the Nrf2 antioxidant response by SAC is strongly associated with its neuroprotective effects against experimental stroke and suggest that targeting the Nrf2 pathway may provide therapeutic benefit for the treatment of stroke. The transcription factor Nrf2 is involved in cerebral ischemic disease and may be a promising target for the treatment of stroke. We provide novel evidence that SAC confers neuroprotection against ischemic stroke by activating the antioxidant Nrf2 signaling pathway. ARE, antioxidant response element; GCLC, glutathione cysteine ligase regulatory subunit; GCLM, glutathione cysteine ligase modulatory subunit; HO-1, heme oxygenase-1; JNK, c-Jun N-terminal kinase; Keap1, Kelch-like ECH-associated protein 1; Maf, musculoaponeurotic fibrosarcoma; Nrf2, nuclear factor erythroid-2-related factor 2; SAC, S-allyl cysteine; ROS, reactive oxygen species.

Novel FTY720-Based Compounds Stimulate Neurotrophin Expression and Phosphatase Activity in Dopaminergic Cells.

α-Synuclein is a chaperone-like protein implicated in Parkinson's disease (PD). Among α-synuclein's normal functions is an ability to bind to and stimulate the activity of the protein phosphatase 2A (PP2A) catalytic subunit in vitro and in vivo. PP2A activity is impaired in PD and in dementia with Lewy Bodies in brain regions harboring α-synuclein aggregates. Using PP2A as the readout, we measured PP2A activity in response to α-synuclein, ceramides, and FTY720, and then on the basis of those results, we created new FTY720 compounds. We then measured the effects of those compounds in dopaminergic cells. In addition to stimulating PP2A, all three compounds stimulated the expression of brain derived neurotrophic factor and protected MN9D cells against tumor-necrosis-factor-α-associated cell death. FTY720-C2 appears to be more potent while FTY720-Mitoxy targets mitochondria. Importantly, FTY720 is already FDA approved for treating multiple sclerosis and is used clinically worldwide. Our findings suggest that FTY720 and our new FTY720-based compounds have considerable potential for treating synucleinopathies such as PD.