Disease‐modifying effects of ganglioside GM1 in Huntington's disease models
2017; Springer Nature; Volume: 9; Issue: 11 Linguagem: Inglês
10.15252/emmm.201707763
ISSN1757-4684
AutoresMelanie Alpaugh, Danny Galleguillos, Juan Forero, Luis Carlos Morales, Sebastian W.K. Lackey, Preeti Kar, Alba Di Pardo, Andrew Holt, Bradley J. Kerr, Kathryn G. Todd, Glen B. Baker, Karim Fouad, Simonetta Sipione,
Tópico(s)Neurological disorders and treatments
ResumoResearch Article9 October 2017Open Access Source DataTransparent process Disease-modifying effects of ganglioside GM1 in Huntington's disease models Melanie Alpaugh Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Danny Galleguillos Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Juan Forero Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Luis Carlos Morales Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Sebastian W Lackey Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Preeti Kar Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Alba Di Pardo Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Andrew Holt Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Bradley J Kerr Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Kathryn G Todd Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Psychiatry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Glen B Baker Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Psychiatry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Karim Fouad Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Simonetta Sipione Corresponding Author [email protected] orcid.org/0000-0002-3060-0723 Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Melanie Alpaugh Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Danny Galleguillos Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Juan Forero Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Luis Carlos Morales Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Sebastian W Lackey Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Preeti Kar Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Alba Di Pardo Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Andrew Holt Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Bradley J Kerr Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Kathryn G Todd Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Psychiatry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Glen B Baker Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Department of Psychiatry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Karim Fouad Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Simonetta Sipione Corresponding Author [email protected] orcid.org/0000-0002-3060-0723 Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Author Information Melanie Alpaugh1,2, Danny Galleguillos1,2, Juan Forero2,3, Luis Carlos Morales1, Sebastian W Lackey1, Preeti Kar1, Alba Di Pardo1,†, Andrew Holt1, Bradley J Kerr2,4, Kathryn G Todd2,5, Glen B Baker2,5, Karim Fouad2,3 and Simonetta Sipione *,1,2 1Department of Pharmacology, University of Alberta, Edmonton, AB, Canada 2Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada 3Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, AB, Canada 4Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, AB, Canada 5Department of Psychiatry, University of Alberta, Edmonton, AB, Canada †Present address: Center for Neurogenetics and Rare Diseases, IRCCS Neuromed, Pozzilli, Italy *Corresponding author. Tel: +1 780 492 5885; E-mail: [email protected] EMBO Mol Med (2017)9:1537-1557https://doi.org/10.15252/emmm.201707763 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by motor, cognitive and psychiatric problems. Previous studies indicated that levels of brain gangliosides are lower than normal in HD models and that administration of exogenous ganglioside GM1 corrects motor dysfunction in the YAC128 mouse model of HD. In this study, we provide evidence that intraventricular administration of GM1 has profound disease-modifying effects across HD mouse models with different genetic background. GM1 administration results in decreased levels of mutant huntingtin, the protein that causes HD, and in a wide array of beneficial effects that include changes in levels of DARPP32, ferritin, Iba1 and GFAP, modulation of dopamine and serotonin metabolism, and restoration of normal levels of glutamate, GABA, L-Ser and D-Ser. Treatment with GM1 slows down neurodegeneration, white matter atrophy and body weight loss in R6/2 mice. Motor functions are significantly improved in R6/2 mice and restored to normal in Q140 mice, including gait abnormalities that are often resistant to treatments. Psychiatric-like and cognitive dysfunctions are also ameliorated by GM1 administration in Q140 and YAC128 mice. The widespread benefits of GM1 administration, at molecular, cellular and behavioural levels, indicate that this ganglioside has strong therapeutic and disease-modifying potential in HD. Synopsis Huntington's disease (HD) is an incurable neurodegenerative disease. Intraventricular administration of ganglioside GM1 in three different HD mouse models ameliorates huntingtin levels, brain and striatal atrophy, motor and cognitive defects and other neurochemical levels. Administration of GM1 decreases levels of toxic soluble and insoluble mutant huntingtin. GM1 slows down neurodegeneration and white matter atrophy in R6/2 mice. GM1 restores normal levels of key neurotransmitters in the brain. GM1 improves and even restores to normal motor function in HD mice, and corrects psychiatric-like and cognitive dysfunction. GM1 could be a novel disease-modifying therapy for HD. Introduction Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the pathological expansion of a trinucleotide (CAG) repeat in the gene that codes for huntingtin (HTT) (Group THsDCR 1993). This mutation results in an abnormally long poly-glutamine stretch at the N-terminus of the mutant HTT (mHTT) protein, which in turn causes mHTT to misfold into toxic species and to aggregate. A plethora of molecular, cellular and network dysfunctions ensue, eventually leading to neuronal death (Imarisio et al, 2008). Cerebral cortex and corpus striatum are the most affected brain regions (Vonsattel et al, 2011), while more subtle pathological changes occur in other brain areas (Petersen & Bjorkqvist, 2006; Aziz et al, 2009; Vonsattel et al, 2011). Motor dysfunction is the hallmark of HD and defines disease onset (Roos, 2010). However, cognitive and psychiatric problems often precede the appearance of motor symptoms and are frequently the most distressing for patients and their families (Paulsen et al, 2008; Roos, 2010). To date, there is no cure or disease-modifying therapy for HD. Clinical management of HD symptoms is possible to a certain extent with the use of tetrabenazine to reduce chorea, and with traditional antidepressant and anti-psychotic drugs (Ross & Tabrizi, 2011). However, the use of these symptomatic treatments is often limited by their potential side effects (Rosenblatt, 2007; Killoran & Biglan, 2014), and the overall efficacy of antidepressants in HD patients is controversial (Moulton et al, 2014). Furthermore, none of these treatments target the underlying causes of dysfunction nor are able to slow down HD progression. In previous studies, we showed that the synthesis of gangliosides—sialic acid-containing glycosphingolipids—is affected in cellular and animal models of HD (Desplats et al, 2007; Denny et al, 2010; Maglione et al, 2010), resulting in lower levels of ganglioside GM1 and, to a lesser extent, other major brain gangliosides (Maglione et al, 2010). Gangliosides perform a plethora of modulatory functions in cell signalling, cell–cell interactions and calcium homeostasis (Sonnino et al, 2007; Posse de Chaves & Sipione, 2010; Ledeen & Wu, 2015). Their importance in the central nervous system is underscored by the fact that knockout mouse models that lack complex gangliosides undergo neurodegeneration (Proia, 2003) and display motor impairment (Chiavegatto et al, 2000), depression-like behaviour (Wang et al, 2014) and learning and memory deficits (Sha et al, 2014). This suggests that reduced GM1 levels in HD may contribute to disease pathogenesis and/or progression. In support of this hypothesis, we showed that administration of exogenous GM1 decreases HD cell susceptibility to apoptosis in vitro (Maglione et al, 2010) and corrects motor dysfunction in the YAC128 mouse model (Di Pardo et al, 2012). However, whether the treatment was able to ameliorate other important aspects of the disease—including non-motor symptoms—and to attenuate the underlying molecular dysfunctions and neurodegeneration remained to be investigated. In this study, we used three different—and for many aspects complementary—HD mouse models (R6/2, Q140 and YAC128 mice) (Mangiarini et al, 1996; Menalled et al, 2003; Slow et al, 2003) to show that chronic intraventricular infusion of GM1 has profound disease-modifying effects that include a reduction in brain levels of toxic mHTT, restoration of normal brain concentrations of specific neurotransmitters, as well as decreased neurodegeneration and white matter atrophy, among others. These changes are accompanied by improvement or even restoration of motor function and by correction of behavioural abnormalities related to depression, anxiety and cognition. Results Intracerebroventricular infusion of GM1 slows down neurodegeneration, improves neuropathology and decreases body weight loss in R6/2 mice To determine the effects of GM1 on HD brain neuropathology, we performed chronic intracerebral infusion of this ganglioside in R6/2 mice (Appendix Fig S1). Compared to other models used in this study, R6/2 mice present with an accelerated disease phenotype and develop widespread neurodegeneration from a young age (Mangiarini et al, 1996). Treatment with GM1 significantly attenuated striatal atrophy (effect of treatment: F1,40 = 152.4, P < 0.0001—see also Appendix Table S1 for P-values relative to pairwise comparisons; Fig 1A) and loss of striatal neurons (effect of treatment: F1,35 = 21.0, P < 0.001) compared to vehicle (artificial cerebro-spinal fluid, CSF)-treated R6/2 mice (Fig 1B). Brain volume (between bregma 1.98 and −2.3; Fig 1C) and total brain weight (Fig 1D) were also significantly higher in R6/2 mice treated with GM1 compared to vehicle-treated mice (effect of treatment: F1,41 = 14.33, P < 0.001; effect on brain volume: P = 0.0092). While brain weight decreased progressively in R6/2 mice from 6 to 10 weeks of age (effect of age: F3,33 = 10.86, P < 0.0001), administration of GM1 for 4 weeks significantly slowed down this process, maintaining brain weight at levels observed at 8 weeks of age without treatment (P = 0.99). Altogether, these data suggest that treatment with GM1 significantly slowed down neurodegeneration in R6/2 mice. Figure 1. GM1 decreases neuropathology and weight loss in R6/2 miceSix-week-old mice were infused with GM1 or artificial cerebro-spinal fluid (CSF, vehicle) for 28 days. Analysis was performed at the end of treatment (10 weeks of age). Striatal volume in the brain left hemisphere (LH, contralateral to infusion site). N = 13 WT CSF, 11 WT GM1, 11 R6/2 CSF, 9 R6/2 GM1. Number of neurons (NeuN+ cells) in the LH striatum between bregma 0.02 mm and −2.3 mm. N = 8 WT CSF, 5 WT GM1, 7 R6/2 CSF, 6 R6/2 GM1. Volume of the brain (LH) from bregma 2.1 mm to −2.3 mm. N = 13 WT CSF, 11 WT GM1, 11 R6/2 CSF, 9 R6/2 GM1. Time course of brain weight loss in R6/2 mice. N = 3 6-week R6/2, 10 8-week R6/2, 14 10-week R6/2 CSF and 10 10-week R6/2 GM1. Corpus callosum volume (LH) between bregma 2.1 mm and 0.02 mm. N = 13 WT CSF, 11 WT GM1, 11 R6/2 CSF, 9 R6/2 GM1. Total white matter tract volume in the striatum, from 0.02 mm to bregma to −1.06 mm. N = 13 WT CSF, 11 WT GM1, 11 R6/2 CSF, 9 R6/2 GM1. Representative microscopy images of the striatum after immunostaining with anti-ferritin antibodies. Scale bars are 0.62 mm in length. Quantification of the immunoreactive area is shown in the graph. Eight serial sections were analysed and averaged for each mouse. N = 3 WT CSF, 4 WT GM1, 6 R6/2 CSF, 5 R6/2 GM1. Percent change in body weight at day 21 of treatment compared to baseline (day 0). N = 23 WT CSF, 21 WT GM1, 14 R6/2 CSF, 11 R6/2 GM1. Survival curve for R6/2 mice treated with CSF (N = 6) or GM1 (N = 5). X-axis shows days after the beginning of GM1 treatment. The horizontal red line indicates the duration of GM1 treatment. Data information: Box-and-whisker plots show median, maximum and minimum values. Two-way ANOVA with Holm–Sidak post-test was used in (A–C and E–H); one-way ANOVA with Tukey's post-test in (D); log-rank analysis was used in (I). *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint GM1 treatment resulted also in decreased atrophy of the corpus callosum (Fig 1E) (interaction: F1,42 = 4.26, P = 0.0451; GM1-treated R6/2 vs. WT: P = 0.9553) and of cortico-striatal white matter tracts (Fig 1F) (effect of genotype: F1,43 = 54.01, P < 0.0001; R6/2 CSF vs. GM1: P = 0.0473) in R6/2 mice that received the ganglioside compared to CSF-treated R6/2 mice. Changes in these white matter structures predate neuronal death and the onset of motor symptoms in HD patients (Paulsen et al, 2006; Jech et al, 2007; Tabrizi et al, 2011, 2012), and correlate with cognitive (Crawford et al, 2013; Novak et al, 2014; Matsui et al, 2015) and cortico-striatal functional deficits (Sapp et al, 1999; Wolf et al, 2008; Douaud et al, 2009), respectively. Dysregulation of iron metabolism and increased ferritin expression are pathological changes that occur in HD patients (Bartzokis et al, 2007b) and R6/2 mice (Simmons et al, 2007) and are likely linked to oxidative stress (Chen et al, 2013a) and dystrophic microglia (Simmons et al, 2007). Upon treatment with GM1, ferritin levels in the brain of R6/2 mice were significantly decreased (interaction: F1,14 = 14.49, P = 0.0019) (Fig 1G). Survival and body weight are frequently used as indices of treatment efficacy in R6/2 mice (Li et al, 2005). Body weight loss was significantly attenuated by administration of GM1 (interaction: F1,65 = 6.43, P < 0.01; Fig 1H). This was not due to increased food intake, which was not significantly different among groups (Appendix Fig S2). Furthermore, GM1 treatment did not affect the weight of WT mice (P = 0.553), suggesting that weight increase in R6/2 mice is the result of overall improved health conditions. R6/2 mice receiving GM1 also showed a trend towards increased lifespan, although these results were not statistically significant (P = 0.10; Fig 1I). GM1 attenuates pathological cellular and molecular changes in HD mice Because of the increasingly recognized role of non-neuronal cell populations in HD pathogenesis (Lobsiger & Cleveland, 2007; Ehrlich, 2012), we next measured the effects of GM1 on astrocytic and microglial markers that are affected by neuroinflammation and in HD (Singhrao et al, 1999; Laurine et al, 2003; Stack et al, 2006; Dalrymple et al, 2007; Giampa et al, 2010; Politis et al, 2011; Baune, 2015; Olejniczak et al, 2015). Immunohistochemistry (Fig 2A and B) and protein immunoblotting (Fig 2C) showed similar levels of glial fibrillary acid protein (GFAP) immunoreactivity in the striatum of WT and R6/2 mice, regardless of treatment. In cortical sections from both CSF- and GM1-treated R6/2 mice, the area covered by GFAP immunoreactivity was slightly higher than in WT littermates (effect of genotype, F1,22 = 4.32, P = 0.049; Fig 2B). In spite of this, GFAP protein expression measured by immunoblotting of cortical lysates was significantly decreased in CSF-treated R6/2 mice compared to WT (effect of genotype: F1,24 = 13, P = 0.0014; Fig 2C). This apparent discrepancy between immunohistochemistry and protein immunoblotting data (i.e. decreased protein expression of GFAP in spite of a larger area covered by GFAP+ astrocytes in cortical tissue) might reflect the coexistence of quantitative as well as qualitative changes in HD astroglia. Administration of GM1 restored GFAP protein expression to WT levels (P = 0.028; Fig 2C). Figure 2. Effects of GM1 on astroglial and microglial markersR6/2 and WT mice were treated with artificial cerebro-spinal fluid (CSF, vehicle) or GM1 for 28 days. A. Representative brain section staining with anti-GFAP antibodies. Areas shown are in the corpus striatum. Scale bars are 50 μm in length. B. Graphs show the quantification of GFAP-immunoreactive area in micrographs of coronal serial sections. For each mouse, eight serial sections were analysed and averaged. N = 11 WT CSF, 9 WT GM1, 10 R6/2 CSF, 8 R6/2 GM1. C. GFAP protein expression in tissue lysates. Representative immunoblots and densitometric analysis are shown. N = 7 WT CSF, 7 WT GM1, 7 R6/2 CSF, 7 R6/2 GM1. The immunoblot showing α-tubulin in the cortex is the same as for the cortex in (F), since GFAP and Iba1 were run in the same gel. D, E. Representative micrographs (D) (from the striatum) and quantification (E) of Iba1+ cell density in the cortex and striatum. For each mouse, eight serial sections were analysed and averaged. Scale bars are 50 μm in length. N = 11 WT CSF, 10 WT GM1, 10 R6/2 CSF, 9 R6/2 GM1. F. Iba1 protein expression in tissue lysates. Representative immunoblots and densitometric analysis are shown. The immunoblot showing α-tubulin in the cortex is the same as for the cortex in (C), since GFAP and Iba1 were run in the same gel. N = 7 WT CSF, 7 WT GM1, 6 R6/2 CSF, 7 R6/2 GM1. G. Spi-1 gene expression in the striatum, analysed by qPCR and normalized over the geometric mean of three stably expressed reference genes. N = 3 WT CSF, 3 WT GM1, 5 R6/2 CSF, 4 R6/2 GM1. Data information: Box-and-whisker plots show median, maximum and minimum values. Two-way ANOVA with Holm–Sidak post-test. *P < 0.05; **P < 0.01. Source data are available online for this figure. Source Data for Figure 2 [emmm201707763-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Similar effects of GM1 were observed on the microglial marker Iba1. Although the number of Iba1+ cells was similar in the cortex of R6/2 and WT mice (P = 0.69) and only slightly decreased in R6/2 striatum (P = 0.03; Fig 2E), unexpectedly Iba1 protein expression was lower than normal in cortical tissue from R6/2 mice (effect of genotype: F1,23 = 20.62, P = 0.001) (Fig 2F). A similar trend was also observed in striatal tissue from R6/2 mice, although here statistical significance was not reached (P = 0.10). GM1 treatment had genotype-dependent effects, decreasing Iba1 protein levels in the striatum of WT animals, but increasing them to control levels in R6/2 mice (interaction: F1,24 = 13.32, P = 0.001; Fig 2F). Like in the case of GFAP expression, our data suggest qualitative differences in the molecular signature of R6/2 microglia that were at least in part mitigated by administration of GM1. In support of this conclusion, expression of the gene encoding the transcription factor Spi-1, a master regulator of microglia biogenesis and function (Heinz et al, 2010; Kierdorf et al, 2013), was lower in vehicle-treated R6/2 mice than in WT animals, and restored to WT levels by GM1 (Fig 2G). Our data also suggest the absence of an overt neuroinflammatory microglia phenotype in our R6/2 mice and experimental conditions. In line with these data, brain levels of major inflammatory cytokines were similar (and low) across genotype and treatment groups (Appendix Fig S3). Next, we determined the effects of GM1 on DARPP32, a key regulator of dopamine (DA) signalling and striatum output pathways (Greengard et al, 1999; Svenningsson et al, 2004). Downregulation—as well as decreased phosphorylation—of DARPP32 marks early dysfunction of HD medium spiny neurons (Bibb et al, 2000). For the analysis of DARPP32 levels (as for GFAP analysis), we only used male mice, to avoid potential confounding effects of the oestrous cycle in female animals (Bode et al, 2008; Hajos, 2008). Chronic treatment with GM1 for 42 days increased expression of DARPP32 and pDARPP32 to WT levels in heterozygous Q7/140 mice (for DARPP32: F4,65 = 23.88, P < 0.0001; Q7/140 CSF vs. GM1 P < 0.001; Q7/7 vs. Q7/140 GM1, P = 0.71. For pDARPP32: F4,65 = 23.2, P < 0.0001; Q7/140 CSF vs. GM1 P < 0.001; Q7/7 vs. Q7/140 GM1, P = 0.93) (Fig EV1). This suggests that GM1 has overall beneficial effects on HD medium spiny neurons signalling and functionality. These specific effects, however, did not extend to homozygous Q140/140 mice, for reasons that are currently unknown (see Discussion). Click here to expand this figure. Figure EV1. GM1 increases DARPP32 and its phosphorylation in Q7/Q140 miceRepresentative immunoblots and densitometric analysis of DARPP32 expression and phospo-Thr34 DARPP32 (p-DARPP32) after treatment with GM1. N = 20 Q7/7 CSF, 13 Q7/140 CSF, 9 Q7/140 GM1, 14 Q140/140 CSF, 15 Q140/140 GM1. Box-and-whisker plots show median, maximum and minimum values. One-way ANOVA with Tukey's post-test. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Mutant HTT levels are reduced by administration of GM1 Mutant HTT is arguably a primary therapeutic target in HD. GM1 treatment caused a reduction of mHTT protein levels in the striatum of both Q7/140 and Q140/140 mice (effect of treatment: F1,11 = 8.798, P = 0.012; HD CSF vs. HD GM1 P = 0.023) after 42 days of treatment (Fig 3A; but not yet after 28 days—data not shown). Since Htt mRNA expression was not affected by GM1 (P = 0.45; Fig 3B), decreased mHTT levels were likely due to increased protein clearance. Wild-type HTT was not significantly affected by the treatment (P = 0.95). Figure 3. Mutant HTT protein levels are reduced by administration of GM1 A. Representative immunoblots and densitometric analysis of wtHTT and mHTT in striatal tissue lysates from Q140 mice after 42 days of treatment with artificial cerebro-spinal fluid (CSF, vehicle) or GM1. Swift™ total protein staining was used as loading control and for data normalization. Homozygous and heterozygous Q140 mice were pooled for the analysis of mHTT as GM1 had similar effects in both genotypes. For the box-and-whisker plot: N = 9 Q140 CSF and 7 Q140 GM1. B. Analysis of total HTT mRNA expression in CSF- and GM1-treated Q140 mice. Data from heterozygous Q7/140 and homozygous Q140/140 mice were similar and were combined. N = 18 Q140 CSF, 17 Q140 GM1. C, D. Filter-trap assay for mHTT insoluble aggregates in tissue lysates. Representative immunoblots and densitometric analysis are shown. N = 12 Q140/140 CSF, 11 Q140/140 GM1, five R6/2 CSF, six R6/2 GM1. SDS-insoluble mHTT aggregates were detected with the indicated anti-HTT antibodies. Only the densitometric analysis for N18 immunoreactivity is shown. Data information: Box-and-whisker plots show median, maximum and minimum values. Two-tailed Student's t-test (A, B) and Mann–Whitney test (C, D). *P < 0.05, **P < 0.01. Download figure Download PowerPoint GM1 also decreased accumulation of SDS-insoluble aggregates of mHTT in the striatum of Q140/140 mice (P = 0.005; Fig 3C). A similar effect was observed in the cortex (P = 0.001), although not in the striatum, of GM1-treated R6/2 mice (Fig 3D). In heterozygous Q7/140 mice, a low amount of SDS-insoluble aggregates and high inter-animal variability likely prevented us from detecting any significant effect of GM1 treatment (data not shown). Treatment with GM1 improves motor performance in R6/2 and Q140 mice The beneficial action of GM1 on HD neuropathology in R6/2 and Q140 mice correlated with amelioration of motor dysfunction in both animal models (Figs 4 and 5), in line with our previous observations in YAC128 mice (Di Pardo et al, 2012). Figure 4. GM1 improves motor behaviour in R6/2 and Q140 miceMotor testing was performed during treatment with cerebro-spinal fluid (CSF, vehicle) or GM1, between day 7 and day 21 of treatment for R6/2 mice, and after day 28 of treatment for Q140 mice. A, B. Horizontal ladder test. Total error score from five consecutive passes is shown. N = 24 WT CSF, 21 WT GM1, 20 R6/2 CSF, 18 R6/2 GM1; N = 26 Q7/7 CSF, 23 Q140 CSF, 26 Q140 GM1. C, D. Open field activity test. The distance travelled during 5-min-long sessions is reported. For Q140 mice, the distance travelled relative to Q7/7 is shown. N = 23 WT CSF, 21 WT GM1, 20 R6/2 CSF, 17 R6/2 GM1; N = 27 Q7/7 CSF, 30 Q140 CSF, 22 Q140 GM1. E. Climbing test. Number of rears performed in 5 min of placement in a wire mesh container. N = 25 Q7/7 CSF, 29 Q140 CSF, 27 Q140 GM1. F. Fixed-speed (12 RPM) rotarod test. Latency to fall is the average of three consecutive trials for each animal. N = 14 Q7/7 CSF, 15 Q140 CSF, 9 Q140 GM1, all females. Data information: Box-and-whisker plots show median, maximum and minimum values. Statistical analysis in (A, C) was performed using a linear mixed effect regression model with 95% confidence intervals calculated at each time point; asterisks indicate statistically significant differences between HD CSF and HD GM1. For all other data, one-way ANOVA with Bonferroni correction was used. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Figure 5. GM1 corrects gait abnormalities in Q140 mice A. Representative stick diagram decompositions (5 ms between sticks) of the left iliac crest and toe motion for a Q7/7 CSF mouse, a Q140 CSF mouse and a Q140 GM1 mouse during walking on a walkway. B. Mean (± s.e.m.) values for iliac crest height. N = 9 Q7/7 CSF (five males, four females), 17 Q140 CSF (seven males, 10 females), 18 Q140 GM1 (eight males, 10 females). C. Mean (± s.e.m.) values for stride duration. N = 9 Q7/7 CSF (five males, four females), 17 Q140 CSF (seven males, 10 females), 18 Q140 GM1 (eight males, 10 females). D–F. Footfall diagrams obtained from video analysis of locomotion were used to calculate: (E) stance-to-stride ratios and (F) coupling phase values (homologous LF/LH and RF/RH, homolateral RH/LH and RF/LF and diagonal LF/RH and RF/LH). Mean (± s.e.m.) values. N = 9 Q7/7 CSF (five males, four females), 17 Q140 CSF (seven males, 10 females), 18 Q140 GM1 (eight males, 10 females). Data information: Statistics were performed using separate three-factor ANOVAs (GROUP [3] × SEX [2] × PUMP [2]) to determine whether there was an effect of the sex (SEX), the side at which the pump hang (PUMP) and the treatment group (GROUP) on the different kinematic measures. Significant differences (P < 0.05) are indicated by asterisks (*) for main effects, and by section signs (§, pump hanging on the right) and daggers (†, pump hanging on the left) for interaction effects. Download figure Download PowerPoint In the horizontal ladder test, a measure of skilled motor control (Metz & Whishaw, 2002), GM1 significantly improved per
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