Anle138b modulates α‐synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy

ABSTRACT Background MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α‐synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP‐hαSyn mouse model expressing human α‐synuclein in oligodendrocytes. At present, there is no effective disease‐modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies. Objectives To test the therapeutic potential of anle138b in a mouse model of MSA. Methods Two‐month‐old PLP‐hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP‐hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed. Results We observed a reversal of motor function to healthy control levels when PLP‐hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α‐synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α‐synuclein reduction observed. Conclusions Anle138b reduces α‐synuclein accumulation in PLP‐hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

synucleinopathies, characterized by misfolding and accumulation of α-synuclein (α-syn). [3][4][5] In MSA, α-syn accumulates in the cytoplasm of oligodendrocytes, forming the so-called glial cytoplasmic inclusions (GCIs), thus differing from PD where α-syn mainly accumulates in neurons in Lewy bodies. [3][4][5] α-syn accumulation in MSA leads to glial and neuronal dysfunction, neuroinflammation and finally neurodegeneration. 1 MSA is divided in two different subtypes depending on the main areas affected by neurodegeneration. The Parkinson's variant or MSA-P is characterized by striatonigral degeneration (SND), whereas the cerebellar variant or MSA-C reflects olivopontocerebellar atrophy (OPCA). 1 The PLP-hαSyn transgenic mouse model recapitulates most of the clinical and pathophysiological features of MSA by overexpressing human α-syn under the oligodendrocyte PLP (myelin proteolipid protein) promoter, which leads to GCI formation, microglial activation, and selective neurodegeneration. [6][7][8][9][10][11][12][13][14][15][16] Microglial activation and neuroinflammation constitute important pathological features of MSA. [17][18][19] Similarly to the human pathology, PLP-hαSyn mice develop progressive microglial activation, initially triggered by α-syn pathology in a region-specific manner. 13,15 In a recent publication from our group, significant microglial activation was observed in the SN of MSA transgenic mice compared to healthy control animals at 5 months of age, and this activation was linked to abnormal neuroinflammatory response. 15 PLP-hαSyn mice also develop progressive SND characterized by a 30% reduction in the number of dopaminergic neurons (tyrosine hydroxylase positive [TH + ] neurons) in the SNc compared to healthy control animals. This neuronal loss is already present at 4 months of age and is followed by a significant reduction in density of dopaminergic terminals and in number of medium spiny neurons in the striatum at 12 months of age. 15,20 SN and striatum are both essential for motor control, and the loss of neurons in these two brain areas leads to motor impairment in these mice. 12,15 According to several studies, under physiological conditions α-syn is mainly located in the pre-synaptic terminals of the neurons predominantly as a monomer. 21 Although its function is still unknown, a possible role in neurotransmitter release, synaptic function, and plasticity has been suggested. 22,23 Misfolding, oligomerization, and aggregation of α-syn are crucial events in the pathophysiology of synucleinopathies. 24 In MSA, the origin of α-syn inclusions in oligodendrocytes is still unknown and under discussion given that it is not clear whether or not mature oligodendrocytes express α-syn. [25][26][27] In the last years, several publications have shown the ability of α-syn to be transferred from cell to cell and spread through brain parenchyma in a prionlike manner. [28][29][30][31][32][33] Based on this, possible neuronoligodendrocyte transfer of α-syn has been suggested in addition to the controversial oligodendrocytic origin of the misfolded α-syn. 34 Several studies in the last few years have shown that α-syn oligomers constitute the main neurotoxic species for disease progression instead of large fibrillar deposits or inclusions. [35][36][37][38][39] According to all these studies, the inhibition of α-syn oligomerization constitutes a promising approach to fight the spreading of synucleinopathies and an important effort has been made in this direction. 21,40,41 The use of small molecules to target α-syn oligomerization and aggregation has shown promising results in preclinical models of PD as is the case of anle138b, a small compound with high bioavailability and low toxicity. 42 This compound can be delivered orally and penetrates the blood-brain barrier, entering the brain with high efficacy. 42 Thus, administration of anle138b as a food additive results in adequate and stable drug exposure. 43 Anle138b has been shown effective in reducing disease progression in models of PD, prion disease, tauopathy, and Alzheimer's disease (AD) by inhibiting protein aggregation. [42][43][44][45] Based on this, we hypothesized that it could also be of use to attenuate disease progression in MSA. In order to test this hypothesis, we fed PLP-hαSyn transgenic MSA mice with food pellets containing two different doses of anle138b followed by behavioral and histological analyses at the end of the treatment.

Animals and Treatments
PLP-hαSyn mice overexpressing wild-type (WT) human α-syn under the PLP promoter, an oligodendroglialspecific promoter, and generated in a C57/BL6 background 16 were used in this study. Animals were kept under temperature-controlled, pathogen-free conditions on a light/dark 12-hour cycle. All the experiments were performed according to the ethical guidelines with the permission of the Austrian Federal Ministry of Science and Research (permission BMFWF-66.011/0141-WF/ v/3b/2016). Two-month-old male transgenic mice were randomized in three different groups: one fed with placebo food pellets (n = 10; ssniff Spezialdiäten GmbH, Soest, Germany), another fed with pellets containing anle138b at 0.6 g/kg of food (n = 8; ssniff Spezialdiäten GmbH), and a last group fed with pellets containing 2 g of anle138b per kg of food (n = 10; ssniff Spezialdiäten GmbH). The dose of 2 g of anle138b per kg of food was used in previous experiments in mice 43 and establishes during the wake phase a concentration of 60 μM in the brain. Two-monthold C57/BL6 healthy nontransgenic animals (WT) fed with placebo pellets were used as a healthy control (n = 10). Food pellets were provided to the animals throughout the whole experiment. After 4 months of treatment, behavioral analyses were performed followed by sacrifice of the animals and brain extraction.

Challenging Beam Test
At the end of the treatment, animal cages were randomly numbered by the animal caretaker and behavioral analyses were performed by the researcher blinded to genotype and treatment. Motor performance and coordination were analyzed with a modified version of the traditional beam test adapted from a previously published method. 46,47 Five performances were video recorded per animal, and the number of slips per step with the hind limbs was measured. The best three performances were used for statistical analyses.

Tissue Processing and Histology
After behavioral analyses were performed, animals were perfused intracardially with phosphate buffered saline (PBS [pH 7.4]; Sigma-Aldrich, St. Louis, MO) under deep thiopental anesthesia and brains were extracted. For molecular analyses, brains were snap frozen in liquid nitrogen and stored at -80 C. For histological analyses, brains were postfixed overnight in 4% paraformaldehyde (pH 7.4; Sigma-Aldrich) at 4 C. After fixation, brains were washed in PBS and then transferred to 30% sucrose (in PBS) until they sank. Finally, brains were frozen using 2-methylbutan (Sigma-Aldrich) and stored at -80 C for further analyses. In order to perform histological analyses, brains were serially cut in 40-μm-thick coronal sections using a freezing microtome (Leica Microsystems, Wetzlar, Germany) and stored free-floating in a cryoprotectant buffer at -20 C. One series was directly mounted on slides and stained with cresyl violet.

Image Analyses
After immunostaining, and preceding the acquisition of images/stereological counting, all immunofluorescence/immunohistochemistry slides were randomly numbered by the laboratory technician. All measurements were therefore performed by the researcher blinded to genotype and treatment. Neuroanatomy was assessed using a Mouse Brain Atlas. Stereological analysis was performed using the Nikon E-800 microscope equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, Japan) and Stereoinvestigator software (Microbrightfield Europe E.k., Magdeburg, Germany) as described previously. 48 GCI density was assessed by meander scan throughout the area of interest and expressed in number of GCI/mm 2 . The number of TH + and cresyl violet positive (CV + ) neurons in the SNc was measured by applying the optical fractionator workflow. For microglial activation assessment, images were acquired with a fluorescence microscope (Leica DMI4000; Leica Microsystems), and the CD68-positive area was estimated using ImageJ software (National Institutes of Health, Bethesda, MD). Results are presented as percentage of CD68 area per SN area.

Continuous Sucrose-Gradient Assay
For continuous gradient centrifugation, a 10% (w/v) brain homogenate of the midbrain was prepared using a buffer composed of 50 mM of Tris (pH 7.4), 175 mM of NaCl, 1 mM of MgCl 2 , 0.1 mM of phenylmethylsulfonyl fluoride, 1 mM of N-ethylmaleimide, 0.1% Nonidet P-40 Substitute, ethylenediaminetetraacetic acid-free cOmplete Mini protease inhibitor (Roche, Indianapolis, IN), and PhosSTOP phosphatase inhibitor (Roche). Aliquots of the homogenate were frozen in liquid N 2 and stored at -80 C. Total protein concentration was determined by bicinchoninic acid assay. For the sucrose gradient analysis, 300 μg of protein in a final volume of 200 μL were used. To this end, the brain homogenate was thawed on ice and diluted in a buffer containing 50 mM of Tris (pH 7.4), 175 mM of NaCl, 0.1% N-lauroylsarcosine sodium salt (sarcosyl), and 0.5% sodium deoxycholate. Samples were agitated at 1,200 rpm (ThermoMixer C, Eppendorf ) at 4 C for 30 minutes. Cellular debris was removed afterward by centrifugation for 1 minute at 16.000 g and 4 C. Sucrose gradients were prepared in a 4-mL 11 × 60 mm polyallomer tube (Beckman Coulter, Brea, CA). To this end, sucrose solutions containing 50 mM of Tris (pH 7.4), 0.1% sarcosyl, and 10%, 20%, 30%, 40%, 50%, or 60% of sucrose were pipetted into the tube, starting with 200 μL of the 60% sucrose solution and followed by 400 μL of the 50% to 10% sucrose solutions. Two hundred microliters of the centrifugation supernatant was pipetted as the uppermost layer onto the gradient. Samples were ultracentrifuged in a SW 60 Ti rotor (Beckman Coulter) at 40,000 rpm and 4 C for 1 hour. Twelve fractions of 200 μL each were collected from the top to the bottom of each tube and subjected to trichloroacetic acid (TCA) precipitation (10% TCA) overnight at -20 C. After thawing, samples were centrifuged at 25,000 g and 4 C for 15 minutes. Precipitates were washed once with acetone (-20 C), centrifuged, and finally resuspended in Laemmli sample buffer. Samples were boiled at 96 C for 5 minutes and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. Antibodies against total α-syn (4B12; BioLegend, San Diego, CA) and phosphorylated α-syn (pS129; Abcam, Cambridge, MA) were used to stain the blots. Images were acquired using the Fusion FX system for western blot and gel imaging, and quantified with FUSION CAPT software (V16.09b; Vilber Lourmat Sté, Collégien, France).

Statistical Analyses
All statistical analyses were conducted using the software Graph-Pad Prism (version 7; GraphPad Software Inc., La Jolla, CA). Mean AE standard error of the mean (SEM) was used to present the results. One-way analysis of variance (ANOVA) with a post-hoc Bonferroni test was used to compare groups if not indicated otherwise. A P value <0.05 was considered statistically significant. Correlations were studied using linear regression analysis.

Anle138b Prevents Motor Deficits and Neurodegeneration in the PLP-hαSyn Mice
To assess the effect of anle138b on motor function, motor coordination and balance were analyzed using the beam challenging test 46,47 (Fig. 1). PLP-hαSyn mice fed with placebo pellets showed a significant increase in the number of slips per step when traversing the beam compared to healthy control animals (Fig. 1). However, PLP-hαSyn mice treated with both doses of anle138b maintained normal motor function as compared to healthy control mice (Fig. 1).
To evaluate whether the improvement in motor function was related to a neuroprotective effect of anle138b on dopaminergic neurons in the SNc, the number of TH + cells was quantified in this region. Stereological counting showed a clear preservation of dopaminergic neurons in the SNc of PLP-hαSyn mice treated with both doses of anle138b compared to healthy controls ( Fig. 2A,B). PLP-hαSyn mice fed with placebo pellets, however, showed a significant loss of TH + neurons compared to all groups ( Fig. 2A,B). The neuroprotective effect of anle138b in SNc was validated by cresyl violet staining followed by stereological counting of neurons in this brain area (Supporting Information Fig. S1A). There was a significant correlation between the neuroprotective effect of anle138b and improvement in motor function ( Fig. 2C and Supporting Information Fig. S1B).

Anle138b Reduces α-syn Accumulation in PLP-hαSyn Mice
Next, the effect of anle138b on α-syn aggregation and intracellular accumulation was assessed. Brain sections of PLP-hαSyn mice were immunostained by using antibodies against α-syn, and the number of GCIs per area in striatum and SN was analyzed. Both doses of anle138b showed a significant effect on GCI number in SNc and striatum, where a 30% reduction was observed (Fig. 3A-C and Supporting Information Fig. S2A,B). This effect was confirmed with the different antibodies used in the study (Fig. 3A-C and Supporting Information  Fig. S2A,B). There was a significant correlation between GCI reduction and preservation of neurons in SNc ( Fig. 3D and Supporting Information Fig. S1C,D and S2C) as well as motor function (Fig. 3E and Supporting Information Fig. S2D).
In addition to the effect on the amount of GCIs, sucrose-gradient analysis showed a significant reduction of α-syn oligomers in midbrains of PLP-hαSyn mice treated with anle138b (Fig. 3F,G). This reduction was observed not only in total α-syn oligomers, but also in pathological phosphorylated α-syn oligomers (Fig. 3F, G), thus confirming the modulatory effect of the small molecule on α-syn oligomerization.

Anle138b Reduces Microglial Activation in PLP-hαSyn Mice
To assess the effect of anle138b treatment on microglial activation, we performed immunofluorescence microscopy for CD68, a lysosomal marker indicative of phagocytic activity of microglia 49 associated with α-syn accumulation. 15,[50][51][52] In agreement with previous results, 15 significant microglial activation was observed at this stage in the SN of the PLP-hαSyn placebo group compared to healthy control animals (Fig. 4A,B). In contrast, both doses of anle138b significantly reduced microglial activation in the SN back to its normal levels as observed in healthy control mice (Fig. 4A,B). There was a significant correlation between the reduction of microglial activation in SN, GCI reduction ( Fig. 4C and Supporting Information Fig. S2E), preservation of neurons ( Fig. 4D and Supporting Information Fig. S1E), as well as motor function (Fig. 4E).

Discussion
Aggregation and accumulation of misfolded proteins constitute a key factor in the progression of several neurodegenerative diseases. Thus, the use of compounds or therapies capable of reducing or blocking abnormal protein aggregation represents a promising strategy to attenuate the clinical progression in these disorders. Synucleinopathies are pathologically characterized by the abnormal aggregation and accumulation of α-syn, and, in recent years, different approaches have been developed to target this process. [3][4][5]21,40,41 The use of small molecules and immunotherapy constitute two of the main strategies with therapeutic potential according to preclinical in vivo and in vitro models. 53,54 Small molecules present important advantages such as their high oral bioavailability and blood-brain barrier penetration and their ability to act inside cells. 53 Here we show that anle138b, which targets specifically oligomers, reduces α-syn accumulation in a mouse model of early-stage MSA, thus corroborating the oligomer modulation effect on α-syn previously observed in PD models and the general effect observed also in Abeta, tau, and prion aggregation mouse models. [42][43][44][45] An important advantage of this small molecule, from a oligomers is blocked, also less α-syn fibrils are generated. In accord with this, our data show that oral administration of anle138b reduces α-syn oligomers in midbrains of PLP-hαSyn mice and leads to a 30% reduction in the number of GCI in the striatum and SN of MSA mice, two brain areas associated with motor function. We also demonstrate that the reduction of α-syn accumulation prevents loss of dopaminergic nigral neurons and motor impairment, thus confirming the therapeutic effect of anle138b observed also in PD models. 42,44 Finally, our data show that the decrease of α-syn aggregation in the SN is associated with a reduction of microglial activation back to the levels observed in healthy control animals in this brain region, which also correlates with the neuroprotective effect of anle138b on dopaminergic cells and preservation of motor function.
The results presented here demonstrate that anle138b treatment is beneficial in early-stage MSA as modeled by young PLP-hαSyn mice. 15 However, efficacy of anle138b was less prominent in a model of pathologically advanced MSA. 60 In that study, the potential of anle138b was evaluated in 1-year-old PLP-hαSyn mice treated with the mitochondrial toxin, 3-nitropropionic acid (3-NP), to trigger full-blown MSA-like pathology, with spreading of GCI, SND, and OPCA and strong microglial activation. 12 Behavioral analyses showed significant motor improvement after short-term treatment with anle138b; however, no significant changes in α-syn aggregate load or cell death were observed in these animals. 60 The variable efficacy of anle138b in the two studies may be attributed to short (1 month) versus long-term (4 months) treatment, but also reflect the fact that PLP-hαSyn animals had sustained different degrees of neurodegeneration in the absence or presence of 3-NP exposure. Moreover, the limited efficacy of anle138b in our first experiment may have been attributed to the addition of toxin-induced acute oxidative stress that cannot be counteracted by the administration of anle138b, given that it has no antioxidant properties. In contrast, to clarify the antiaggregation and neuroprotective potential of anle138b, a different, preventive approach was used in the present study, where chronic administration of the compound started before nigral degeneration of dopaminergic neurons occurred and in the absence of other non-αSyn-dependent deleterious stimuli.
In summary, our study shows that oral administration of anle138b with initiation in the early disease stages constitutes a promising approach to prevent disease progression in MSA (Fig. 5). Our observations and previous studies have demonstrated the potential of anle138b in different neurodegenerative disorders, and the data presented here support further development of anle138b for future clinical applications in patients suffering from MSA or related synucleinopathies.