Preclinical Validation of FTY720 and FTY720-Mitoxy in Mouse Models of Parkinsons Disease and Multiple System Atrophy (MSA): Evidence for Treating Lewy Body Disease Synucleinopathies Including MSA

Abstract

We assessed FTY720 and our patented-mitochondria-localizing-FTY720-derivative, FTY720-Mitoxy, in mouse models of Parkinson’s disease (PD) and MSA. FTY720 and FTY720-Mitoxy were given by gavage, injection, or osmotic pump. We used symptomatic transgenic alpha-Synuclein (aSyn) PD mice (A53T aSyn) and MSA mice (CNP-aSyn), as well as transgenic GM2 +/− PD mice. We also tested toxin PD and MSA models. We measured movement, constipation, gut motility, sweat ability, and bladder function. We counted blood lymphocytes 24 h after FTY720 or FTY720-Mitoxy. We measured Brain Derived Neurotrophic Factor (BDNF), Glial Cell Line Derived Neurotrophic Factor (GDNF), and Nerve Growth Factor (NGF) mRNA and protein. We assessed aSyn insolubility in gut, brain, and spinal cord by sequential protein extraction and immunoblot. We assessed fecal genomic DNA using 16S rRNA sequencing. In PD mice FTY720 normalized body and gut movement, urinary bladder function while increasing trophic factors and eliminating synucleinopathy. In MSA mice FTY720-Mitoxy normalized body and gut movement, sweat ability, mitochondrial function, improved microbiota while increasing trophic factors and eliminating synucleinopathy. FTY720 and FTY720-Mitoxy improve function and counteract synucleinopathy. As FTY720-Mitoxy is not immunosuppressive, it may be safer for treating PD and/or MSA.

1. Introduction

PD and MSA are progressive neurodegenerative disorders with overlapping movement and autonomic symptoms that typically manifest over time during the aging process. Fortunately, there are mouse models that manifest symptoms of both diseases which allows for modeling them for drug testing. PD and MSA are both what are called “synucleinopathies” because they occur after the small chaperone-like protein, alpha-synuclein (aSyn), accumulates primarily inside neurons in PD and glial cells in MSA, leading to brain and body dysfunction [1, 2]. But one may wonder, why does the aSyn aggregation induce mainly neuronal problems in PD [3] and glial abnormalities followed by neuronal loss in MSA [4, 5]?

One clue about how this happens arises from data showing that PD motor impairment occurs only after a marked loss of substantia nigra dopaminergic neurons [6, 7], while surviving nigral neurons typically contain intracellular aSyn aggregates known as Lewy bodies (LBs) [8]. Only rare PD cases are associated with aSyn mutations and/or multiplications that are causative of the disease [9]. Recent evidence has shown that there are different conformationally and biologically distinct strains of pathological aSyn in PD and MSA [10]. Regardless of its strong association with PD and MSA, it is also very well documented that aSyn has important normal functions, some of which are especially vital for dopaminergic neurons where aSyn interacts with key proteins that modulate not only dopamine synthesis [11, 12] but also dopamine release [13, 14]. This modulation occurs, at least in part, because aSyn binds to and stimulates the activity of protein phosphatase 2A (PP2A), a major Ser/Thr phosphatase that regulates the phosphorylation state of the enzyme tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and also key proteins that modulate cell signaling [15, 16]. Significantly, in vivo silencing of aSyn expression in mature nigrostriatal neurons in vivo causes neuronal dysfunction and subsequent brain neuroinflammation to occur [17]. Additionally, when aSyn aggregates in human brains and tissues from animal models, dysregulation of PP2A activity occurs [18] which can lead to an excess of intracellular and extracellular dopamine, reactive oxygen species, and dopamine quinones, which are highly toxic to the nigral dopaminergic neurons [19, 20, 21]. Furthermore, another major enzyme, monoamine oxidase B, which helps degrade dopamine, is modulated by interaction with aSyn [22]. Together these data confirm that aSyn plays a key role in regulating brain pathways that control normal body movement.

In MSA, aSyn accumulates inside the myelinating glial cells of brain, the oligodendrocytes, which then forms two different types of lesions (1) glial cytoplasmic inclusions (GCI) and (2) glial nuclear inclusions (GNI) [23, 24, 25]. When this occurs, neuronal demyelination occurs along with a loss of trophic factors required to support neurons, ultimately impairing function [26, 27, 28]. MSA can manifest as two different forms, (1) a condition that primarily affects the basal ganglia, thus producing Parkinsonian symptoms (MSA-P) or (2) primarily as cerebellar dysfunction and ataxia (MSA-C) [27]. Because transgenic PD and MSA mouse models recapitulate many of the age-onset changes associated with PD and MSA [29, 30, 31, 32, 33, 34, 35] they have been used to assess potential therapies [36, 37, 38, 39, 40, 41, 42, 43]. While many therapeutics for PD and MSA are in development [44, 45], the focus of this review is on our preclinical validation of FTY720 (along with data from other laboratories who studied FTY720), and our patented derivative, FTY720-Mitoxy, as possible therapies for PD and MSA.

Because many symptoms of PD and MSA arise over time in humans and can be modeled in aging mice, drugs can be evaluated over time for their ability to slow, halt, or reverse PD or MSA symptoms. For the purposes of review in this chapter, we focus on the symptoms that are labeled in red font and underlined in Figure 1.

Figure 1.

PD and MSA both manifest after the onset of motor symptoms (listed on the left) and/or nonmotor symptoms (listed on the right), with symptoms in red and underlined, occurring in aging PD and MSA mouse models. Figure created with BioRender.com.

To test the efficacy of FTY720 and FTY720-Mitoxy as potential therapies for PD and MSA we, and others, evaluated several mouse models and tested multiple methods. Though this chapter is a review of our published data, we included an abbreviated Materials and Methods section below (Section 2) to help more clearly summarize all of the models and methods employed over many years of study, in order to simplify data interpretation.

2. Materials and methods

2.1 Mice as models of PD and MSA

Briefly, all of the mice studied in our laboratories were housed in barrier cages on ventilated racks in temperature and humidity-controlled rooms on 12-h light/dark cycles with food and water available ad libitum. Mice underwent behavioral testing in clean quiet test rooms after acclimation as previously described. Ethical treatment of mice followed AALAC, ARRIVE, and NIH guidelines on approved protocols at Texas Tech University Health Sciences Center or at Shandong University as also previously described.

2.2 PD mouse models

2.2.1 A53T aSyn Tg mice

Mice expressing the A53T mutant form of human aSyn were generated in the laboratory of Dr. Virginia Lee (University of Pennsylvania, Philadelphia, PA) [46]. We purchased the heterozygous (B6:C3-Tg-Prnp/SNCA*A53T/83Vle/J) breeders from the Jackson Laboratories (Bar Harbor, ME). Our cohort of Tg mice thus consisted of WT non-transgenic littermates as well as and heterozygous and homozygous offspring that overexpressed either one or two copies of mutant human A53T aSyn driven by a prion promoter [46]. Genotyping was performed as described [47]. Onset of PD-like symptoms were verified prior to drug treatment.

2.2.2 GM2 Tg mice

Mature gangliosides are ligands for myelin associated glycoprotein (MAG) [48] that act to enhance myelin stability [49] and contribute to normal brain function. It has been shown that immature GM3 gangliosides are elevated in PD brain [50], while mature GM1 ganglioside levels are significantly lower in PD patients and in PD mouse models [51, 52]. Transgenic B4galnt1 heterozygous mice, gift of Drs. Ledeen and Wu (Rutgers New Jersey Medical School), were used to generate our colony of wild type mice (WT, +/+) with two normal copies of the GM2 Synthase gene, and heterozygous mice with a single GM2 Synthase gene (GM2, +/−). Genotyping was performed as described [53, 54]. Thus, the GM2+/− heterozygous mice, with excess GM3 and reduced GM1 levels, closely model human PD. Onset of PD-like symptoms were verified prior to drug treatment.

2.2.3 6-OHDA toxin model

6-OHDA has been widely used to model PD in which striatal injections produce nigral dopamine neuron loss over 1–3 weeks’ time, allowing for longitudinal assessment of the mice [55]. Briefly, C57BL/6 mice were pretreated with 0.5 mg/kg FTY720 or vehicle by intraperitoneal injection for 7 days prior to 6-OHDA. On the 7th day, 1 h after final FTY720 dosing, mice were placed in a stereotaxic apparatus under anesthesia and injected with 6 μg of 6-OHDA (prepared in 2 μL of normal saline with 0.02% ascorbic acid) or saline alone into two different sites of the right striatum. All mice were assessed at 7-, 14-, or 21-days post injection [56].

2.2.4 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model

Two different laboratories studied the impact of FTY720 in the MPTP toxin model of PD. Komnig et al. [57], used a subacute dose of MPTP, while Motyl et al. [58] used an acute MPTP treatment to induce PD like changes. Komnig found no benefit with FTY720 while Motyl and colleagues reported significant protection against MPTP toxicity using FTY720.

2.2.5 Rotenone model

Zhao et al. [59], used rotenone to induce PD-like symptoms in their mice, which caused systemic mitochondrial impairment, oxidative damage, microglial activation, selective nigrostriatal dopaminergic degeneration, L-DOPA-responsive motor deficits, aSyn aggregation and formation of Lewy body-like inclusions. FTY720 was reported to be highly protective in this model. Another laboratory tested FTY720-Chitosan against a rotenone model and also saw protection [60].

2.3 MSA mouse models

2.3.1 CNP-aSyn Tg mice

CNP-aSyn mice (B6:C3-Tg-CNP-SNCA-M2Vle) were obtained from a repository established by Dr. Virginia Lee at the Jackson Laboratories (Bar Harbor, ME). Heterozygous, non-littermate Tg mice were used to generate our CNP-aSyn mouse colony. Heterozygous or homozygous Tg mice express either one or two copies of WT human SNCA with expression driven by a CNP promoter in the myelinating cells of CNS (oligodendroglia) and peripheral nervous system (PNS, Schwann cells) [61, 62]. This CNP-aSyn model develops progressive motor and autonomic problems as well as neuronal and white matter damage in response to aSyn overexpression, similar to what occurs in subjects suffering with MSA-P and MSA-C [39, 63]. Onset of MSA-like symptoms was verified prior to drug treatment.

2.3.2 The 3NP toxin model of MSA

MSA models can also be generated using the mitochondrial toxin 3-nitropropionic acid (3NP) (Cat # N5634, Sigma-Aldrich, St. Louis, MO, USA), which inhibits mitochondrial succinate dehydrogenase (SDH) activity, also known as mitochondrial Complex II resulting in MSA like symptoms [64]. We prepared 3NP with sterile saline (pH 7.4) and sterile saline used along as the control. Aging CNP-aSyn mice (8.5 mo) were given subcutaneous injections of 3NP twice daily at escalating doses—10 mg/kg (day 9.5), 20 mg/kg (days 11 and 12), and 30 mg/kg (days 13 and 14) exactly as described [39, 65].

2.4 Drugs evaluated

All FTY720 and FTY720-Mitoxy (Figure 2) used in our studies, were evaluated for purity and stability prior to use [39, 66]. FTY720 is a synthetic sphingosine-1-phosphate receptor modulator approved to treat relapsing remitting multiple sclerosis (MS) [67]. MS patients benefit from FTY720 by both its anti-inflammatory and neuroprotective effects, which we have also demonstrated to occur in response to FTY720-Mitoxy, both in vitro and in vivo [68, 69].

Figure 2.

FTY720 and FTY720-Mitoxy chemical structures. Figure is reprinted with permission of Elsevier Science and Technology Journals, Experimental Neurology [39] from the Copyright Clearance Center.

Notably, both drugs increase the expression of brain derived neurotrophic factor (BDNF), while FTY720-Mitoxy also significantly increases the expression of nerve growth factor (NGF) and glial-cell-line derived neurotrophic factor (GDNF) in neuronal and oligodendroglial cells [39, 53, 68, 70, 71].

The major difference between the drugs is the fact that FTY720, which is phosphorylated in vivo by Sphingosine Kinase, causes immunosuppression. In stark contrast, FTY720-Mitoxy, which also rapidly crosses the blood brain barrier but is not phosphorylated [66] does not suppress the immune system [72].

2.5 Behavioral tests to assess drug efficacy in mice

2.5.1 Movement tests

• Rotarod (Cat # LE8200, Harvard Apparatus, Holliston, MA), measures balance, coordination, and endurance. Timing as “latency to fall” from the apparatus was recorded in sec using established methods [46, 73]. Briefly, mice were familiarized with the rotarod during initial trainings of 2 sessions on 2 consecutive days. Each training session consisted of 2 runs lasting 2 min each, one at 4 revolutions per min (rpm) and the other at 8 rpm. Experimental data were collected in 3 runs/day on 2 different days for each mouse, with rotation increasing from 4 to 40 rpm over 5 min. A minimum 5 min rest period was allowed between runs for all mice [39, 71].

• Open field locomotor activity was monitored using the TruScan™ open field apparatus (Coulbourn Instruments, Whitehall, PA, USA). Mice were acclimated to the test room for 15 min prior to being placed individually in the center of the arena with total movement monitored for 15 min. Total movement was measured as successive coordinate movements made across the floor plane while mice were continuously active. Mice were tested in random order on two independent occasions with established methods [53, 74].

• Apomorphine-induced rotations were monitored over 3 weeks’ time, from 1 week post 6-OHDA lesioning as before [11]. Apomorphine was subcutaneously injected into mice at a dose of 0.1 mg/kg (Sigma), with mice placed individually in plastic beakers (diameter: 13 cm), and videotaped from above for 30 min. Quantitative analyses of completed (360°) left and right rotations were made off-line by an investigator blinded to the experimental conditions.

• Hindlimb reflex tests were performed as follows. Each mouse was suspended by the tail for 5 sec during which the position of the hindlimbs was scored. Data were collected over 3 trials performed on 2 different days, with short breaks given between trials. Scores ranging from impaired to normal were as follows: 0 = one or both hindlimbs paralyzed, 1 = hindlimbs and paws close to the body with clasping toes, 2 = loss of flexion of hindlimbs, 3 = hindlimbs extended <90° angle, and 4 = hindlimbs extended >90° angle.

2.5.2 Gut function and gut health

• Fecal pellets. When food moves through the gut slowly, the colon absorbs more water, so consequently the feces become dryer and hardened. Thus, water content in feces was measured using methods described by Taylor et al., with total stool collected in the afternoon from individual mice placed in clean cages for 1 h. Feces were immediately transferred to 1.5-mL Eppendorf tubes that were labeled, capped, and weighed. Tubes were opened to allow fecal desiccation, and heated at 65°C overnight. Tubes were capped and weighed again, with water content calculated by computing the difference between wet and dry weights [47, 71].

• Gastrointestinal transit time. Whole gut transit time was assessed after oral gavage of a 0.2-mL volume of 6% (w/v) carmine red dye in 0.5% methylcellulose (Sigma-Aldrich). Post-gavage, all mice were observed up to 9 h for excretion of the first red stool, with the time recorded for each mouse. Mice that had not passed any red stool by 9 h scored >9 h [47, 75].

• Colonic motility was measured in old mice using the bead expulsion test. Briefly, a glass bead (3 mm; Sigma-Aldrich, Z143928-1EA) was gently pushed 2.0 cm into the colon using the smooth end of a plastic inoculating loop (Nunc, 253287). The total time from bead insertion to bead ejection was recorded for each mouse [47].

• Microbiota were analyzed by 16S rRNA sequencing at Texas Tech in Lubbock after extracting fecal genomic DNA in our laboratory at Texas Tech in El Paso. Briefly, human subjects collected their own fecal samples at home into a container filled with stool DNA stabilizer (PSP® Spin Stool DNA PlusKit, Stratec Molecular). After transfer to the laboratory, samples were stored at −80°C until processing. Mouse feces were collected into sterile tubes during 1 h periods in the morning, with feces preserved and stored exactly as above. Total fecal DNA was extracted using QIAamp fast DNA stool Mini kits (Cat # 51604, Qiagen Inc., Valencia, CA) followed by 16S rRNA analysis as described [76].

2.5.3 Sweat function

• The starch iodine test was used to assess sweating in mice with MSA-like changes. Sweat droplets were measured using established methods [77] with minor modifications. Specifically, mice were gently and firmly manually restrained by one experimenter throughout each test. Another experimenter cleaned the left hind paw with a water-moistened cotton tipped swab then painted that paw using a small artist brush dipped in a freshly prepared solution of 2% iodine (Cat # 207772, Sigma-Aldrich, St. Louis, MO, USA) in ethanol. After the paw dried, it was then painted with a starch solution prepared in castor oil (1 g/mL, Cat # S9765 in Cat # 259853, Sigma-Aldrich, St. Louis, MO, USA). The paw was then photographed through a 10X magnifier lens at 0.0, 2.5 and 5 min timepoints to record the presence of dark purple precipitates. Digital images were blind coded and the main paw areas (excluding digits) were analyzed using ImageQuant 5.2 (GE Healthcare, Waukesha, WI) to quantify droplets in arbitrary units [39].

2.5.4 Bladder function

• Water intake was confirmed for individually housed mice with water being delivered using a 50 mL conical tube sealed with a #7, single hole rubber stopper and a double-ball water sipper. Tubes were weighed before tests and again the next morning to calculate water intake for all mice [53]. This was done to assure that mice were hydrated for urine pattern tests.

• Urine patterns were assessed as a measure of autonomic function. Collection of urine. Food and water were removed during tests. Five independent tests were conducted per mouse at each time point. Each mouse was individually placed in a clean cage for 1 h (10:00–11:00 am) with the cage bottom covered with a fitted white filter paper (Bio-Rad, Hercules, CA, USA, cat# 1650962). Filter papers were collected, labeled, and dried. Analysis. Urine spots were illuminated by UV light and categorized as small (<0.2 cm²) or large (>0.2 cm²). Counting was done by individuals blinded to genotypes. If overlapping urine spots were detected, those were excluded from counts (Table 1) [53, 71].

Symptom evaluated	Test employed
Postural instability	Hindlimb reflexes
Walking and gait problems	Rotarod, open field, apomorphine induced rotation
Sweat function	Starch iodine
Gastrointestinal (GI) function/GI health	Fecal pellets, GI transit time, bead expulsion, microbiota
Bladder function	Urinary patterns

Table 1.

Summary of symptoms evaluated and tests employed.

2.6 aSyn solubility to measure synucleinopathy

• Sequential protein extraction was performed using tissues from treated mice by well-established methods [18, 78]. This method does not isolate cellular or subcellular fractions but rather soluble from insoluble proteins using a series of buffers and ultracentrifugation with pellet re-extractions. The concentration of protein was confirmed by bicinchoninic acid assay (BCA, Thermo Pierce, Rockford, IL, USA). Laemmli buffer was added to samples, that were then heated to 95°C for 5 min before SDS-PAGE, with the exception of SDS/Urea samples, which were not boiled before gel loading. Proteins were transferred to nitrocellulose, with equivalent protein loading verified by Ponceau S stain.

• Immunoblots. Blots were blocked 1 h in 10% milk-PBS then incubated overnight at 4°C in aSyn antibody (sc-7011R, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), 610786 (BD Biosciences, San Jose, CA, USA). Infrared signal was obtained using anti-mouse, anti-goat, or anti-rabbit secondary antibodies coupled to IgG IRDye680 or IgG IRDye800 (1:5000–1:10,000; Rockland Immunochemical, Boyertown, PA, USA) with blots imaged using Odyssey (LiCor Biosciences, Lincoln, NB, USA).

2.7 Trophic factor expression

• Total mRNA was extracted from brain or paw sweat glands using RNeasy Plus Mini Kit (Cat # 74134, Qiagen Inc., Valencia, CA) followed by retrotranscription with a RNA-to-cDNA Kit (Cat # 4387406, Thermo Fisher Scientific), as per manufacturer. Total miRNA from brain was extracted using miRNeasy Mini Kits (Cat # 217004, Qiagen). Mature miRNAs were retrotranscribed with miScript II RT Kit (Cat # 218160, Qiagen). RNA concentrations and purity were confirmed by NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific). Integrity of RNAs and assessment of genomic DNA contamination were done by evaluating 28S/18S rRNAs band ratios on RNA “bleach” gels as previously described [39].

• BDNF, GDNF, and NGF Protein were assessed on immunoblots loaded with equivalent amounts of total protein for each condition, as determined using the BCA assay as described in Section 2.5.

2.8 Mitochondrial assessment

• Dounce homogenization of 200 mg cerebellum/mouse was performed on ice to isolate brain mitochondria using a kit (Cat # 89801, Thermo Fisher Scientific). Protease inhibitors: 17 μg/mL aprotinin, 1 mM benzamidine, and 1 mM AEBSF were added to all solutions. The final pellet contained mitochondria suspended in 50 μL sterile PBS. Total protein concentrations of isolated mitochondria were determined by BCA as described in Section 2.5.

• Succinate dehydrogenase (SDH) activity was measured using 60 μg of purified mitochondria and a colorimetric assay (Cat # MAK1971KT, Sigma-Aldrich, St. Louis, MO USA), as previously described [39].

3. Results

3.1 Benefits of FTY720 and FTY720-Mitoxy in PD and MSA mouse models

Data from multiple publications are summarized below in tabular and graphic format confirm that FTY720, whose international patent recently expired and is now available in generic form, as well as our patented derivative FTY720-Mitoxy, show that both are extremely protective in PD and MSA mouse models. Both drugs reverse symptoms of abnormal body and gut movement as well as autonomic dysfunction including constipation, abnormal sweating, and abnormal urination. Both drugs also reduce or eliminate synucleinopathy as measured by sequential protein extraction evaluated by aSyn immunoblot (Figure 3 and Table 2).

Figure 3.

Summary of FTY720 and FTY720-Mitoxy data that support taking the necessary next steps to test the safety and efficacy of FTY720 and FTY720-Mitoxy to slow PD or MSA. Top row: drawings and images illustrating tests and variables used to assess body movement, constipation, sweating, incontinence, and aSyn pathology in PD and MSA mouse models. Middle row: models of PD (A53T, GM2 +/−, 6-OHDA) and MSA (CNP-aSyn, 3NP), showing drugs tested in that model, in parentheses. Bottom row: drug effects on body movement, gut/bowel activity, sweat function, bladder function, and ability to sustain soluble aSyn and counteract synucleinopathy. Figure was created using BioRender.com.

Mice studied	Modeling	Variable measured	FTY720	FTY720-Mitoxy	Reference
A53T aSyn	PD	Movement (body/gut)	Protects	NT	[47]
GM2 +/−	PD	Movement (body)	Protects	NT	[53]
		Bladder function	Protects	NT	[53]
Rotenone	PD	Movement (body)	Protects	NT	[59]
MPTP	PD	Movement (body)	Protects	NT	[58]
6-OHDA	PD	Movement (body)	Protects	NT	[56, 59]
CNP aSyn	MSA	Movement (body/gut)	NT	Protects	[39]
3NP	MSA	Mitochondrial SDH	NT	Protects	[39]
Control mice	Normals	Immunosuppression	Positive	Negative	[72]
Rotenone/Chitosan-FTY	PD	aSyn phosphorylation, PP2A modulation	Protects	NT	[60]

Table 2.

Data confirming FTY720 and FTY720-Mitoxy protection in vivo. PD or MSA onset was confirmed before initiating drugs (NT = not tested).

3.1.1 Trophic factors

Both drugs increase the expression of brain derived neurotrophic factor (BDNF), while FTY720-Mitoxy also significantly increases expression of nerve growth factor (NGF) and glial-cell-line derived neurotrophic factor (GDNF) in neurons and oligodendroglia cells [39, 53, 68, 70, 71].

3.1.2 Safety

A major difference between FTY720 and FTY720-Mitoxy is the fact that FTY720 becomes phosphorylated in vivo by Sphingosine Kinases, which leads to its immunosuppressive effects. Having FTY720 act in an immunosuppressive manner, while beneficial for MS patients with that autoimmune disorder, could be problematic for aging individuals suffering with PD or MSA as will be further described below. In stark contrast, FTY720-Mitoxy, which also rapidly crosses the blood brain barrier but is not phosphorylated [66] does not suppress the immune system [72], making it a potentially safer drug for treating patients with PD or MSA aSyn pathology.

3.1.3 Microbiota

We saw an increase in Ruminococcus in all mice after FTY720-Mitoxy, but an increase in Bacteroides only in FTY720-Mitoxy treated CNP-aSyn MSA mice (Figure 4). Ruminococcus is considered beneficial. Bacteroides are considered “friendly commensals” when residing in the gut, where they act to functionally out-compete other bacteria and/or viruses to prevent infection [79]. Because all FTY720-Mitoxy treated CNP-aSyn MSA mice appeared healthy and had improved behavioral functions [39, 76], it is believed that Bacteroides in their gut were also beneficial.

Figure 4.

Data from wild type (WT) control mice and CNP-aSyn MSA littermate mice collected 8–12 weeks post FTY720-Mitoxy treatment. This figure from Vidal-Martinez et al. [76], is re-printed with permission of iospress.nl.

3.1.4 Succinate dehydrogenase (SDH) activity

After FTY720-Mitoxy treatment of 3NP treated MSA mice, we isolated cerebellar mitochondria and measured their SDH activity relative to freshly prepared standards using established protocols. FTY720-Mitoxy significantly enhanced mitochondrial function in both WT and CNP-aSyn MSA 3NP mice. Specifically, 3NP treatment alone reduces SDH activity in WT and CNP-aSyn mitochondria. 3NP + FTY720-Mitoxy double treatment returns SDH activity to normal baseline values in WT and CNP-aSyn mitochondria as did FTY720-Mitoxy in WT mitochondria, but most significantly, FTY720-Mitoxy treatment improved SDH activity in the mitochondria isolated from transgenic CNP-aSyn MSA mice [39].

4. Discussion/conclusions

Herein, we demonstrate robust in vivo protection by FTY720 and FTY720-Mitoxy in several different mouse models of PD and/or MSA. Both drugs significantly slowed or even reversed disease progression while nearly totally counteracting aSyn pathology, also known as synucleinopathy. These are key requirements for drugs that are urgently needed for treating PD and MSA. Notably, FTY720 (fingolimod, Gilenya) could now be used off-label for PD or MSA should a doctor choose to prescribe it. It is worth noting that we used low dose FTY720 when treating our mice, and low dose FTY720 has been made available for treating children with multiple sclerosis. However, a major concern for using FTY720 for PD or MSA still remains because its immunosuppressive effects sometimes induce the opportunistic infection known as progressive multifocal leukoencephalopathy (PML) [73], which can be fatal.

This concern is what encouraged us to create novel FTY720-derivatives, FTY720-C2 and FTY720-Mitoxy, that are not phosphorylated and do not cause immune suppression [72]. And although FTY720-Mitoxy is not an oral drug, it very rapidly enters the brain when given by injection or when delivered over time and it also is highly stable, which allowed long-term delivery (over several weeks). Thus, we have demonstrated the preclinical proof of concept for FTY720-Mitoxy, which like insulin could be given by injection, transdermal patch, or pump to help improve a patient’s quality of life [75].

Our preclinical findings with FTY720-Mitoxy show that it is highly beneficial in MSA models, which supports taking the necessary next steps in drug development to move this patented compound toward the clinic. While ours is currently the only laboratory to have published preclinical data regarding FTY720-Mitoxy, others now have the opportunity to test the drug in their own laboratories, as several vendors now offer FTY720-Mitoxy for sale.

Prior to embarking on Phase I safety or pharmacodynamics studies in healthy human subjects and/or affected subjects, FTY720-Mitoxy requires further characterization to verify its safety and dosing requirements. Importantly, because MSA is an Orphan disorder, with MSA patients often progressing rapidly, such steps to add FTY720-Mitoxy to the arsenal of potential therapies for MSA would be highly beneficial not only for MSA but perhaps also for more common synucleinopathies like PD, dementia with Lewy bodies, and even for Alzheimer’s disease.

5. Patents

The corresponding author holds patents “Compositions and Methods for the Treatment of Parkinson’s Disease” in the USA (#10,391,066) and Canada (#2,888,634) for the FTY720-derivatives, FTY720-C2 and FTY720-Mitoxy.

Acknowledgments

We appreciate support from the Steven Hoy family, Glanville/Reynolds/Perez Families, Anna Mae Doyle, Michael J. Fox Foundation, US National Institutes of Health, NINDS (R01-NS42094), Fogarty International Center (NCOD-5D43TW008333), TTUHSC Graduate Program, Multiple System Atrophy Coalition, Coldwell Foundation, El Paso Community Foundation, Paso Del Norte Foundation (to R.G.P.); Chinese Ministry of Education (BS2010YY036), National Natural Science Foundation (81274124, 81200982) and Shandong Province Science & Technology Program (2014GSF118038, 2016GSF201061) (to H.L.). Funders played no role in study design, data collection, analysis, data interpretation; manuscript writing; or decision about where to publish.

Author contributions

Dr. Perez did most of the writing, Table and Figure preparation. Dr. Vidal-Martinez performed most animal studies, wrote key portions on gut microbiota, and provided essential editorial feedback. Dr. Lou did key mouse experiments and provided editorial feedback. All authors approved the final version before submission.