Brief Communication | Published:

Vascular endothelial growth factor ameliorates the ataxic phenotype in a mouse model of spinocerebellar ataxia type 1

Nature Medicine volume 17, pages 14451447 (2011) | Download Citation


Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by expansion of a glutamine repeat tract in ataxin-1 (ATXN1). Although the precise function of ATXN1 remains elusive, it seems to be involved in transcriptional repression. We find that mutant ATXN1 represses transcription of the neurotrophic and angiogenic factor vascular endothelial growth factor (VEGF). Genetic overexpression or pharmacologic infusion of recombinant VEGF mitigates SCA1 pathogenesis, suggesting a new therapeutic strategy for this disease.


Transcriptional alteration is the earliest pathogenic signature in SCA1 mouse models, suggesting that dysregulated gene expression caused by mutant ATXN1 is central to the pathogenesis of SCA1,2,3,4,5. However, little is known about the direct targets of ATXN1-induced repression, particularly those genes that are dysregulated in the vulnerable Purkinje cell population. Moreover, it is unclear whether correcting the expression of aberrantly expressed genes can ameliorate SCA1.

Because Purkinje cells, the most susceptible neurons in SCA1, constitute less than 0.1% of cerebellar cells, we used laser-capture microdissection (LCM) to enrich for these neurons to identify dysregulated gene expression (Fig. 1a and Supplementary Fig. 1a; see Supplementary Methods for all methodology). We used tissue from SCA1 knock-in mice (henceforth referred to as SCA1 mice), a model that closely mirrors human SCA1 (ref. 6). This SCA1 mouse model expresses an expanded version of ATXN1 with 154 glutamine repeats, in contrast to a non–disease-causing repeat of 2 in mice and less than 40 in humans.

Figure 1: VEGF is downregulated in Purkinje cells of SCA1 mutant mice.
Figure 1

(a) Isolation of mouse cerebellar Purkinje cells by LCM (3-month-old mice). Left, section before LCM, arrow points to the Purkinje cell layer; middle, section after LCM with the Purkinje cell layer cut out; right, pure population of Purkinje cells on the retrieval cap. Scale bar, 50 μm. (b) mRNA expression of Vegfa, Gsbs, Homer3 and Slc1a6 in LCM Purkinje cell samples from SCA1 mice compared to wild-type littermates (normalized to actin). P values using two-tailed t test are as follows: Vegfa, P < 0.001; Gsbs, P < 0.05; Homer3, P < 0.01; and Slc1a6, P < 0.05. Data represent five pairs of SCA1 and wild-type mice. (c) VEGF protein amounts in cerebella of SCA1 knock-in mice compared to wild-type littermates. Each ELISA was done in duplicate and normalized to the weight of cerebella. n = 13 pairs of SCA1 and wild-type littermates 6 month of age or older. P < 0.05; paired two-tailed t test. (d) VEGF staining of Purkinje cells in the cerebella of a 10-month-old SCA1 mouse and wild-type littermates. Scale bar, 25 μm. (e,f) Relative luciferase activity in N2A cells transfected with a reporter construct containing the Vegf promoter and ATXN1-84Q, ATXN1-2Q (e) or ATNX1-84Q(S776A) (f). Western blot showing amounts of transfected ATXN1. Arrow points to the top band representing ATXN1-84Q, whereas the bottom band is ATXN1-2Q. Data are representative of ten independent experiments done in duplicate, P < 0.05 for all ATXN1-overexpressing conditions compared to no ATXN1 (unpaired two-tailed t test) (e) and five independent experiments, each performed in duplicate, P < 0.05 (unpaired two-tailed t test) comparing ATXN1-84Q and ATXN1-84Q(S776A) (f). (g) ChIP on cerebellar lysates with antibody to ATXN1 and quantitative PCR for the Vegfa promoter (normalized to input using IgG as a reference). Data are representative of three independent experiments, P < 0.05 (unpaired two-tailed t test). (h) Histone acetylation at the Vegfa promoter in SCA1 mice compared to wild-type littermates, as determined by ChIP on cerebellar lysates with antibody to acetylated histones (H3ac); quantitative PCR data are normalized to input and total histone H3 (H3total) amounts, using wild-type mice as a reference. Data are representative of three independent experiments, P < 0.05 (unpaired two-tailed t test). (i) Blood vessel density (by collagen IV staining) in cerebella of nine pairs of wild-type and SCA1 mice (older than 10 months), P < 0.01 (paired t test). Data are represented as means ± s.e.m. RLU, Relative luciferase units.

As the yield of RNA from LCM material was insufficient for microarray screening, we used a PCR-based approach to examine the expression of candidate genes involved in key neurodegenerative pathways. One of the genes that we found downregulated in SCA1 mice encodes VEGFA (henceforth called VEGF), an angiogenic and neurotrophic factor implicated in motor neuron disorders7,8,9 (Fig. 1b). In SCA1 mice the levels of Vegfa mRNA were decreased at postnatal day 30 (Supplementary Fig. 1b), well before any behavioral or pathological signs. The decrease in Vegfa mRNA was more pronounced than that of three other genes previously described as downregulated in SCA1: Gsbs, Homer3 and Slc1a6 (refs. 1,2). In the cerebellum, VEGF is widely expressed in neurons, glia and endothelial cells10,11, with robust expression in Purkinje neurons (Fig. 1 and Supplementary Fig. 1c,d).

Low Vegfa mRNA expression was confirmed at the protein level, as Vegf protein abundance was decreased by 30% in SCA1 cerebella as compared with wild-type cerebella (Fig. 1c,d). The decrease in VEGF was most prominent in Purkinje neurons, although granule cells also showed a trend towards lower Vegfa mRNA levels and a significant decrease in Vegf protein abundance (Supplementary Fig. 1e–h). We also found that Vegfa mRNA levels were lower in a transgenic SCA1 mouse model that expresses mutant ATXN1 specifically in Purkinje neurons12 (Supplementary Fig. 1i).

We next asked whether ATXN1 is directly responsible for downregulation of Vegfa mRNA expression. To test whether ATXN1 modulates Vegfa promoter activity, we used a Vegfa luciferase reporter assay13. Both expanded (ATXN1-84Q) and wild-type (ATXN1-2Q) ATXN1 repressed reporter activity in a dose-dependent manner (Fig. 1e). The ability of even wild-type ATXN1 to cause repression is in keeping with the observation that overexpression of wild-type ATXN1 can induce pathology in animal models and the notion that SCA1 can in part be caused by a gain of ATXN1 normal function14. Mutating a phosphorylation site crucial for ATXN1 toxicity (S776A)15 disrupted repression at the Vegfa promoter (Fig. 1f). Thus, ATXN1 repression of the Vegfa promoter correlates with its toxicity in vivo. Consistent with the repression of Vegfa promoter activity, primary cerebellar neurons from SCA1 mice secreted lower amounts of Vegf (Supplementary Fig. 2a). We next performed chromatin immunoprecipitation (ChIP) with an ATXN1-specific antibody to test for ATXN1 occupancy of the Vegfa promoter. The Vegfa promoter (but not the promoter of a closely related family member, Vegfc8) was indeed more abundant in the ChIP lysates (Fig. 1g and Supplementary Fig. 2b). The Vegfa promoter was also hypoacetylated (Fig. 1h), and inhibitors of histone deacetylases relieved ATXN-1 84Q–induced repression at the Vegfa promoter (Supplementary Fig. 2c), suggesting a role for histone acetylation in ATXN1-induced repression.

Given that VEGF is an angiogenic factor, diminished VEGF abundance could contribute to cerebellar dysfunction by limiting angiogenesis. We observed a significant decrease in cerebellar microvessel density and total vessel length in SCA1 mice (Fig. 1i and Supplementary Fig. 3a–d), as well as evidence of hypoxia in SCA1 cerebella using pimonidazole, a hypoxic cell marker (Supplementary Fig. 3e,f).

In addition to its proangiogenic function, VEGF is also a neurotrophic factor. Thus, inadequate VEGF levels could also be deleterious by limiting neurotrophic support to cerebellar neurons. We tested the effects of reduced Vegf amounts on mixed cerebellar neuronal cultures that express Vegf and its receptor Vegfr2 (the predominant VEGF receptor in neurons). Inhibition of Vegf signaling using Vegfr2 tyrosine kinase inhibitors or a neutralizing antibody to Vegf resulted in decreased neurite length and increased cell death (Supplementary Figs. 4–6). Thus, reduced Vegf signaling compromises the growth and survival of not only Purkinje neurons but also other cerebellar neurons through a reduction in neurotrophic support.

We then examined whether restoring VEGF expression improves the SCA1 phenotype in mice. We crossed SCA1 mice with transgenic mice that overexpresses human VEGF in neurons starting at embryonic day 14 (VEGFtg/–; Supplementary Fig. 7a,b and ref. 16). Genetic overexpression of VEGF enhanced motor performance of mice (as assessed by rotarod) at 13 weeks (Fig. 2a) and 6 months (Fig. 2b) of age and improved SCA1 cerebellar pathology (Fig. 2c–e). The thickness of the molecular layer (Fig. 2c), staining intensity of calbindin (which specifically labels Purkinje cells; Fig. 2d,e) and cerebellar microvessel density (Fig. 2f) were increased.

Figure 2: VEGF overexpression improves motor performance and pathological hallmarks in SCA1 mice.
Figure 2

(a,b) Rotarod performance of 13-week-old (a) and 6-month-old (b) SCA1 mice. n = number of mice of each genotype. Data were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. *P < 0.05 (day 1) and **P < 0.001 (days 2, 3 and 4) at 13 weeks. *P < 0.05 for days 1 and 3 and **P < 0.001 for day 4, at 6 months, comparing SCA1 mice to SCA1 mice with one copy of the Vegfa transgene (SCA1;VEGFtg/–). (c,d) SCA1 pathology in 36-week-old mice as determined by the width of the molecular layer (c) and intensity of calbindin staining (d). Data represent one of eight (width) or five (intensity) experiments using independent litters to generate data for four experimental genotypes. Data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. (e) Representative confocal images of 36-week-old mice of the indicated genotypes stained with a calbindin-specific antibody. Scale bar, 40 μm. (f) Cerebella of three quadruplicates of wild-type, SCA1 mice, SCA1;VEGFtg/– and VEGFtg/– (older than 10 months) were examined for the number of blood vessels by collagen IV staining. P < 0.05, determined by one-way ANOVA with Bonferroni post hoc test. Data are represented as means ± s.e.m.

We next tested whether pharmacological delivery of recombinant Vegf is beneficial after disease onset. As Vegf cannot cross the blood-brain barrier, we continuously delivered mouse Vegf using an intracerebroventricular osmotic pump (Supplementary Fig. 7c,d). Intracerebroventricular infusion of Vegf improved motor performance and restored cerebellar pathology (Supplementary Fig. 8).

Our findings suggest a role for VEGF in SCA1 pathogenesis and indicate that restoring VEGF levels may be a potentially useful treatment in patients with SCA1. In addition, these findings advance our understanding of neurodegeneration in SCA1. First, there seems to be cross-talk between the degenerating nervous system and the vascular system, Second, our results may explain the alterations in energy metabolism and decreased oxygen consumption identifiable by functional magnetic resonance imaging in people with SCA1 (ref. 17). Third, alterations in VEGF itself or sequelae of VEGF signaling in blood or cerebrospinal fluid of affected individuals could prove to be a biomarker of disease progression. Finally, because ataxia pathways interact in pathogenic hubs18, our results could prove relevant to other ataxias and possibly even other neurodegenerative syndromes.


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We thank H. Zoghbi (Baylor College of Medicine) and H. Orr (University of Minnesota) for generously providing ATXN1 constructs and the SCA1 mouse models, and S. Leibovich (University of Medicine and Dentistry of New Jersey) and P. D'Amore (Harvard Medical School) for VEGF luciferase reporter constructs. We also thank K. Gobeske for assistance with the intracerebroventricular delivery methods, A. Ma for help with pathological analyses and V. Brandt for editorial assistance. This work was funded by US National Institutes of Health grants K02 NS051340, R21 NS060080 and R01 NS062051 (P.O.); a US National Organization for Rare Disorders grant (P.O.), a US Brain Research Foundation Grant (P.O.) and a US National Ataxia Foundation grant (P.O.). M.C. received funding from the US National Institutes of Health training grant T32. The authors wish to dedicate this manuscript to fellow scientist T. Spann, who is currently fighting amyotrophic lateral sclerosis.

Author information


  1. Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

    • Marija Cvetanovic
    • , Jay M Patel
    •  & Puneet Opal
  2. Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.

    • Hugo H Marti
  3. Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.

    • Ameet R Kini
  4. Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

    • Puneet Opal


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P.O., A.R.K. and M.C. conceived of the study and designed the experiments. M.C. and J.M.P. conducted and analyzed the experiments. H.H.M. provided and characterized the VEGF-transgenic mice. P.O., M.C. and A.R.K. wrote the paper, and J.M.P. helped with revising the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Puneet Opal.

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