Edited by: Tibor Hortobágyi, University of Debrecen, Hungary
Reviewed by: Atsushi Takeda, National Hospital Organization, Sendai-Nishitaga Hospital, Japan; Tibor Kovacs, Semmelweis University, Hungary
*Correspondence: Nadia Stefanova
This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience
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The neurodegenerative disorder multiple system atrophy (MSA) is characterized by autonomic failure, cerebellar ataxia and parkinsonism in any combination associated with predominantly oligodendroglial α-synuclein (α-syn) aggregates (glial cytoplasmic inclusions = GCIs). To date, there is no effective disease modifying therapy. Previous experiments have shown that the aggregation inhibitor anle138b reduces neurodegeneration, as well as behavioral deficits in both transgenic and toxin mouse models of Parkinson's disease (PD). Here we analyzed whether anle138b improves motor skills and reduces neuronal loss, as well as oligodendroglial α-syn aggregation in the PLP-α-syn transgenic mouse challenged with the mitochondrial toxin 3-nitropropionic acid (3-NP) to model full-blown MSA. Following 1 month of treatment with anle138b, MSA mice showed signs of motor improvement affecting stride length, but not pole, grip strength, and beam test performance. Loss of dopaminergic nigral neurons and Purkinje cells was not attenuated and GCI density remained unchanged. These data suggest that the pathology in transgenic PLP-α-syn mice receiving 3-NP might be too advanced to detect significant effects of anle138b treatment on neuronal loss and intracytoplasmic α-syn inclusion bodies. However, the partial motor amelioration may indicate potential efficacy of anle138b treatment that may be mediated by its actions on α-syn oligomers or may reflect improvement of neuronal dysfunction in neural at risk populations. Further studies are required to address the efficacy of anle138b in transgenic α-syn models of early-stage MSA and in the absence of additional toxin application.
Multiple system atrophy (MSA) is a progressive, adult-onset neurodegenerative disorder that belongs to the spectrum of α-synucleinopathies (ASP). MSA patients present with parkinsonism, cerebellar, autonomic, and pyramidal dysfunction in any combination (Fanciulli and Wenning,
In previous work we have replicated MSA-like glial inclusions and mild SND in mice overexpressing human α-syn (hα-syn) under the control of the proteolipid protein (PLP) promoter (Kahle et al.,
All animal experiments were performed according to ethical guidelines and Austrian law as well as with permission from the Federal Ministry of Science and Research, Austria. All efforts were made to reduce the number of animals used and minimize their suffering. All mice were bred and maintained under temperature-controlled, pathogen free conditions, and 12-h light/dark cycle granting free access to water and food at the Animal Facility of Medical University Innsbruck. In the present study a total of 28 homozygous transgenic PLP-α-syn mice, obtained from Prof. Philipp Kahle (Tübingen, Germany) and described previously (Kahle et al.,
Twelve month old homozygous transgenic PLP-α-syn mice were randomized into two treatment groups receiving either vehicle or aggregation inhibitor anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole] treatment. The anle138b group started receiving food pellets (Ssniff, Soest, Germany) that contained the compound anle138b (2 g compound/1 kg food) whereas the control group received food pellets without the compound anle138b 1 week prior to 3-NP intoxication. Body weight of the mice was controlled daily. Food pellets were provided throughout the whole experiment. After 1 week both groups were intoxicated with 3-NP to induce the full-blown pathology similar to human disease (Stefanova et al.,
All behavioral tests were performed by a researcher who was blinded to the treatment status of the animals.
Motor score analysis to assess the severity of 3-NP induced motor disability of the treated transgenic PLP-α-syn mice was performed every day beginning with the first IP 3-NP injection. To estimate hindlimb clasping, general locomotor activity, hindlimb dystonia, truncal dystonia, and postural challenge response a previously described rating scale was applied (0, normal; 1, slightly disturbed; 2, markedly disabled; Fernagut et al.,
The Flex Field Activity System (San Diego Instruments, USA) was used to assess the locomotor activity of the mice upon treatment as previously described (Stefanova et al.,
To test the stride length of the mice after treatment the DigiGait™Analysis System (Mouse Specifics, USA) was used as described elsewhere (Amende et al.,
The pole test has been described as a helpful behavioral test for evaluating movement disorders caused by striatal dopamine depletion (Matsuura et al.,
Fine motor coordination and balance capacities of the mouse were assessed employing the beam walking test (Luong et al.,
To measure forelimb grip strength of mice upon 3-NP injection, the ability to remain clinging to an inverted wire grid for up to 1 min under constant mild shaking was tested in three trials. The best performance was used for statistical analysis.
On day 28 after starting the anle138b treatment, animals were perfused under deep thiopental anesthesia (12 mg/100 g body weight, IP) using phosphate buffered saline (PBS; 25 mM, 0.9% NaCl, pH 7.4) followed by 4% paraformaldehyde (pH 7.4, Sigma-Aldrich, Austria). After removing brains quickly, they were post-fixed in 4% PFA over night at 4°C, and then kept in 30% sucrose (Sigma-Aldrich, Austria) solution until they sank at 4°C. Afterwards, brains were frozen using 2-methylbutan and stored at -80°C until further processing. Whole brains were cut on a freezing microtome (Leica, Nussloch, Germany) processing 40 μm thick sections, whereby one section was collected on slides and six sections were kept free-floating and either dried or kept in assorter buffer.
Immunohistochemical staining was performed according to a standard immunoperoxidase protocol for free-floating sections using following antibodies: mouse anti-dopamine- and cAMP-regulated phosphoprotein (DARPP-32; 1:5000; BD Biosciences, USA), mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000; Sigma, USA), rat anti-human α-syn (aa 116-131 hα-syn; dilution and company please; 15G7, Enzo Life Sciences, Germany), rabbit anti-phosphorylated α-syn (pα-syn; 1:1000; Abcam, UK), and mouse anti-nitrated α-syn (nα-syn; 1:1000; Invitrogen, Zymed Laboratories, USA). In case of the visualization of partially proteinase-K (PK)-resistant human α-syn aggregates, tissue slices were pre-stained with haematoxylin and digested with PK before the incubation with antibodies against α-syn (Neumann et al.,
Stereological analysis was performed by an unbiased investigator at the Nikon E-800 microscope equipped with Nikon digital camera DXM 1200 and Stereoinvestigator Software (MicroBrightField Europe e.K., Germany) as described previously (Mayhew and Gundersen,
All statistical analyses were conducted using the software Graph-Pad Prism 5 (Graphpad Software, San Diego, CA). The mean ± S.E.M was used to present the results. Unpaired
Body weight of vehicle and anle138b treated PLP-α-syn mice was monitored throughout the experiment. No significant differences in body weight between the groups were detected throughout the study. Upon IP injections of 3-NP a typical reduction of body weight was measured with the lowest recordings at the end of 3-NP administration. After day 16 a mild increase followed by a stabilization of the body weight was observed (Figure
General locomotor activity in the vertical (rearing) and horizontal plane in an open field arena showed no significant differences between vehicle- and anle138b-treated PLP-α-syn mice (Figures
Loss of TH-immunoreactive neurons in SNc was induced by systemic treatment with 3-NP in PLP-α-syn mice as shown by the low total number of stereologically estimated TH-positive neurons (Figure
Oral treatment with the compound anle138b had no significant effect on the number of PK-resistant GCIs in any of the regions studied as compared to vehicle-treated mice. Independent of the anle138b treatment, region-specific differences were detectable between SN (SNc and SNr) and cerebellum [CB, stratum granulosum and stratum pukinjense;
As MSA is a fatal primary oligodendrogliopathy associated with GCIs and selective multisystem neurodegeneration novel therapeutic drugs that attenuate brain pathology are of great importance. In the current study we investigated for the first time the effect of the novel aggregation inhibitor anle138b on behavioral outcome, neurodegeneration and α-syn aggregates in MSA mice exposed to mitochondrial inhibition. We observed improvement of stride length variability in anle138b treated MSA mice. However, there were no significant effects on loss of dopaminergic neurons or Purkinje cells and on GCI density in SN and CB.
Promising results exist regarding the reduction of neurodegeneration and pathological deposition of α-syn in different PD mouse models (rotenone, MPTP, and neuronal overexpression of α-syn) upon anle138b administration (Wagner et al.,
One reason for the limited improvement in the current study compared to earlier studies, could be that the compound anle138b was either tested in synucleinopathy models created with toxins OR neuronal overexpression of α-syn, but never in a dual-hit mouse model (Wagner et al.,
Of note, we were not able to identify changes in α-syn inclusion bodies in oligodendrocytes in the MSA model after 1 month of treatment with anle138b, although the drug has been shown previously to counteract α-syn aggregation. As already mentioned we cannot exclude that the duration and the dose of the therapy in this experiment have been insufficient to provide measurable changes in the GCI density and further studies will be needed to address this issue as well as potential effects on α-syn oligomers. Furthermore, in a recent study, 3-NP was shown to impair the autophagic flux in oligodendroglial cells treated with exogenously added α-syn
Comparing human PD to MSA, the latter seems to be the more progressive neurodegenerative disease with a fatal outcome after ~9 years whereas PD patients have a better prognosis (Schrag et al.,
In conclusion, we here report for the first time the effects of the new aggregation inhibitor anle138b in a MSA mouse model combining oligodendroglial α-syn overexpression with 3-NP-induced oxidative stress. Taken together, the data suggest that anle138b induced some improvement of motor impairment supporting further development of the compound for MSA. However, no significant protection of neuronal cells or reduction of oligodendroglial α-syn aggregation was found. Advanced MSA pathology reproduced in the dual-hit mouse model might have been too severe to permit significant neuroprotection with anle138b treatment in the applied dose and duration. Furthermore, interference between oxidative stress and clearance mechanisms of α-syn may have masked the effects of anle138b treatment on oligodendroglial α-syn aggregation in the double-hit model of MSA. Future studies will need to address all these issues to resolve the efficacy of anle138b for the therapy of MSA.
LF—acquisition, analysis, and interpretation of data; drafting and revising the manuscript; final approval of the submission; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. DK—acquisition of data; revising the manuscript; final approval of the submission; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. JL, SR, AL, and CG—interpretation of data; revising the manuscript; final approval of the submission; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. AG, GW, and NS—conception and design; interpretation of data; revising the manuscript; final approval of the submission; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.
AG and CG are co-founders of MODAG. AL is partly employed by MODAG. Remaining co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by grants of the Austrian Science Fund (FWF) W1206, P25161, and F4414, grant of the European Community's Seventh Framework Programme (FP7/2007-2013) under agreement n 603646 (Multisyn).