Neuroprotection by Hsp104 and Hsp27 in Lentiviral-based Rat Models of Huntington's Disease

© The American Society of Gene Therapy

original article

Neuroprotection by Hsp104 and Hsp27 in Lentiviral-based Rat Models of Huntington’s Disease Valérie Perrin1, Etienne Régulier1, Toufik Abbas-Terki1,2, Raymonde Hassig3, Emmanuel Brouillet3, Patrick Aebischer1, Ruth Luthi-Carter1 and Nicole Déglon1,3 Ecole Polytechnique Fédérale de Lausanne (EPFL), Brain Mind Institute, Lausanne, Switzerland; 2Current address: Apotech Corporation, Epalinges, Switzerland; 3Atomic Energy Commission (CEA), Institute of Biomedical Imaging and MIRCen Program, Orsay, France 1

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by an expansion of glutamine repeats in the huntingtin (htt) protein. Abnormal protein folding and the accumulation of mutated htt are hallmarks of HD neuropathology. Heat-shock proteins (hsps), which refold denatured proteins, might therefore mitigate HD. We show here that hsp104 and hsp27 ­rescue striatal dysfunction in primary neuronal cultures and HD rat models based on lentiviral-mediated overexpression of a mutated htt fragment. In primary rat striatal ­ cultures, production of hsp104 or hsp27 with htt171-82Q restored neuronal nuclei (NeuN)–positive cell density to that measured after infection with ­vector expressing the wild-type htt fragment (htt171-19Q). In vivo, both chaperones significantly reduced mutatedhtt-related loss of DARPP-32 expression. Furthermore, hsps affected the distribution and size of htt inclusions, with the density of neuritic aggregates being remarkably increased in striatal neurons overexpressing hsps. We also found that htt171-82Q induced the up-­regulation of endogenous hsp70 that was co-localized with htt inclusions, and that the overexpression of hsp104 and hsp27 modified the subcellular localization of hsp70 that became cytoplasmic. Finally, hsp104 induced the production of endogenous hsp27. These data demonstrate the protective effects of chaperones in mammalian ­models of HD. Received 7 September 2006; accepted 13 February 2007; published online 20 March 2007. doi:10.1038/mt. sj.6300141

INTRODUCTION Huntington’s disease (HD) is a genetic, autosomal-dominant neurodegenerative disease that manifests in midlife and causes involuntary choreic movements and psychiatric and cognitive impairments.1 The HD mutation has been identified as a CAG expansion at the 5′ end of the Htt gene, which is translated into a polyglutamine (polyQ) repeat tract in the huntingtin (htt)

protein. HD neuropathology is characterized by selective degeneration of the GABAergic spiny projection neurons of the striatum and the presence of intracellular htt aggregates in various areas of the brain.2 Misfolded protein typically accumulates in neurons, and this feature is common to several other neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, Creutzfeldt–Jakob disease, and other polyQ diseases.3 However, it remains unclear whether monomers, oligomers, protofibrils, fibrils, or other protein assemblies are the toxic species in these diseases. In HD, inclusions are found in the nucleus or the somatodendritic compartment of neurons and are often immunoreactive for ubiquitin and chaperones.2 These inclusions contain mutated htt but also transcription factors, cytoskeletal proteins, protein kinases, and components of the ubiquitin-proteasome system.4 Chaperones represent one of the cellular defense mechanisms against misfolded proteins. Molecular chaperones, or heatshock proteins (hsps), are a large family of proteins subclassified by molecular size, cellular compartment, and function. Hsps were first identified as proteins rapidly produced in response to stress and involved in protein refolding. It is now acknowledged that chaperones play essential roles in various cellular functions, including the folding of newly translated proteins under ­normal conditions, vesicle fusion, autophagy, signal transduction, apoptosis, and proteasomal degradation.5 Chaperones may prevent polyQ toxicity by facilitating disease protein degradation/­ sequestration or by blocking inappropriate protein interactions but also by altering the downstream signaling events that lead to neuronal dysfunction and cell death. Several chaperone proteins have been reported to play a role in polyQ disorders.6 The overexpression of hsp70/40 decreases aggregate formation in vitro and prevents neurodegeneration in vivo.7–12 In transgenic mice with polyQ disorders, hsp ovexpression has given variable results. Little or no effect on aggregate load and polyQ toxicity was reported in R6/2 and SCA7 mice, whereas very strong hsp70 overexpression was found to abolish neuropathological features and to improve motor function in SCA1 mice.13–15 Recent studies have shown that yeast

Correspondence: Nicole Déglon, Commissariat à l’Energie Atomique (CEA), Service Hospitalier Frédéric Joliot, 4 Place du Général Leclerc, 91401 Orsay Cedex, France. E-mail: [email protected] Molecular Therapy vol. 15 no. 5, 903–911 may 2007

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Figure 1 In vitro expression of heat-shock (hsp) transgenes. (a) E16 rat striatal neurons infected with a lentiviral vector encoding yeast hsp104. Staining with an anti-hsp104 antibody demonstrating strong transgene expression 2 weeks after infection. (b) Higher magnification showing strong hsp104 expression in the entire cell body. (c) Non-infected control culture stained with an anti-hsp104 antibody. (d) E16 rat striatal neurons infected with a lentiviral vector encoding rat hsp27. Staining with an anti-hsp27 antibody demonstrating strong transgene expression 2 weeks after infection. (e) Higher magnification showing strong hsp27 expression in the cytoplasm. (f) Non-infected control cultures stained an anti-hsp27 antibody. Scale bars (b) and (e) 20 µm.

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The effects of hsp104 and hsp27 on HD neuropathology were studied in vitro in a previously characterized primary striatal neuron model of the disease.23–25 This model shows HD-like neurotoxicity between 2 and 8 weeks in vitro, including the appearance of htt- and EM48-positive inclusions, dysmorphic neurites, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling–­positive cell death. Figure 1 shows the expression of hsp transgenes in the E16 rat striatal cultures 2 weeks after infection. Hsp104 was detected in the entire body of infected

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RESULTS hsp104 and hsp27 prevent neuronal dysfunction and cell death in vitro

cells (Figure 1a and b), whereas a diffuse, cytoplasmic staining was observed for hsp27 (Figure 1d and e). The specificity of the immunostaining was demonstrated by comparisons with non-infected cultures (Figure 1c and f). As previously reported,23 the expression of a mutated fragment of htt (htt171-82Q) resulted in a 50% reduction of neuronal nuclei (NeuN)–positive cells at 8 weeks (100.4 ± 1.7 cells per 10 fields; Figure 2d and g) compared with cultures infected with a vector expressing the wildtype htt fragment (htt171-19Q) (210.8 ± 4.0 cells per 10 fields;

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hsp104 counteracts polyQ aggregation and toxicity through its unique ability to promote the refolding of aggregated protein in a two-step process requiring the small hsps (shsps) hsp26 and hsp70/40.16,17 Hsp104 reduces the number of htt inclusions and increases survival in yeast, Caenorhabditis elegans, and mammalian cell models of HD.18–21 Finally, Wyttenbach et al. have demonstrated that hsp27 probably exerts its neuroprotective effects in vitro not through its chaperone function but instead by limiting the production of reactive oxygen species and regulating apoptosis through effects on caspase activation.22 Chaperones clearly reduce polyQ toxicity in yeast and in vitro systems, but studies in rodent models of HD are required to confirm the protective effects and mode of action of these proteins. The unique ability of hsp104 to induce the refolding of already aggregated proteins and the apoptotic or anti-oxidant functions of hsp27 renders them particularly attractive in an HD context. We therefore assessed the effects of these chaperones in both in vitro and in vivo rodent models of the pathology.

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Figure 2  Neuroprotective effects of chaperones in an in vitro model of Huntington’s disease. E16 rat primary striatal cultures were coinfected on day 1 with lentiviral vectors encoding wild-type (htt171 with 19Q repeats) or mutated huntingtin (htt) fragments (htt171 with 82Q repeats) and with vectors encoding hsp104 or hsp27. (a–f) Long-term survival and polyglutamine-induced pathological effects on the striatum were assessed with the neuronal marker neuronal nuclei (NeuN). Eight weeks after infection, the percentage of NeuN-positive cells was significantly lower in cultures infected with the htt171-82Q vector (d) than in cultures infected with htt171-19Q (a). The overexpression of hsp104 (e) or hsp27 (f) prevents the loss of NeuN-positive cell bodies in htt17182Q-infected cultures. The expression of htt171-19Q with hsp104 (b) or hsp27 (c) has no effect on the number of NeuN-positive cells. (g) Quantitative analysis of NeuN-positive cells (N = 6, mean ± SEM, ***P < 0.001). One-way analysis of variance, F(5, 66) = 66.54, P < 0.001. Post hoc comparison of 82Q with 82Q + hsp104 or 82Q + hsp27, ***P < 0.001. Post hoc comparison of 19Q with 19Q + hsp104 or 19Q + hsp27, not significant. (h) The overexpression of mutated htt171-82Q leads to the formation of nuclear (V) and non-nuclear inclusions (*) in 8-week-old rat striatal cultures. (i, j) The expression of htt171-82Q with hsp104 (i, k) leads to an increase in non-nuclear EM48-positive inclusions, whereas the overall ratio of nuclear to non-nuclear EM48-positive inclusions remain unchanged for hsp27 (j, k). (k) Quantitative analysis of average difference between nuclear and non-nuclear inclusions for each group (data are mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001). t-test between nuclear and non-nuclear inclusions for htt17182Q (t stat = 3.94, **P < 0.01), hsp104 (t stat = –2.24, *P < 0.05), and hsp27 (t stat = 6.09, ***P < 0.001).

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hsp104 and hsp27 reduce mutated htt toxicity and modify the distribution of htt inclusions To examine the protective effects of chaperones in vivo, we used a lentiviral-based rat model of mutated htt neurotoxicity.26­ 82Q + hsp104

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Figure 2a and g). PolyQ toxicity was significantly reduced when hsp104 or hsp27 was produced together with htt171-82Q (Figure 2e–g), whereas overexpression of either chaperone with htt171-19Q had no effect on the number of NeuN-­positive cells (Figure 2b, c and g). We assessed the impact of hsps on htt inclusions by staining for htt with the EM48 antibody (Figure 2h–j). In htt171-82Q-infected cultures, EM48-positive inclusions were detected in the nucleus and neurites of infected neurons (Figure 2h). The proportions of non-nuclear and nuclear inclusions were quantified on EM48-propidium iodide doublestained sections (Figure 2h–k). Compared with the htt17182Q group, the co-expression of htt171-82Q and hsp27 has no major impact on the formation and distribution of htt aggregates, whereas a significant increase in the proportion of nonnuclear htt aggregates was observed in hsp104-treated cultures (Figure 2k). At 8 weeks, the number of EM48-positive neurons was lower in cultures infected with htt171-82Q alone, owing to polyQ-mediated toxicity. These results suggest that hsp104 and hsp27 protect rat primary striatal neurons from polyQ insult without reducing the formation of htt EM48-positive inclusion bodies.

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Figure 3  In vivo transgene expression. (a) Experimental design. Lentiviral vectors encoding hsp104 and hsp27 were injected into both sides of the striatum of adult rats (n = 16 per group). One month later, lentiviral vectors expressing htt171-19/82Q were injected into the striatum at the same coordinates (19Q on the left and 82Q on the right). As a control, ten animals were injected with lentiviral vectors expressing htt171-19/82Q alone. As a control for heat-shock protein (hsp) expression, PBS–BSA was injected into the left hemisphere in eight animals from the chaperone groups. The animals were killed 2 months after infection with htt171-19/82Q vectors. (b–e) Huntingtin (htt) expression in htt171-19Q- and htt171-82Q-injected animals was analyzed with the 2B4 antibody 8 weeks after injection. Diffuse cytoplasmic staining was observed for wild-type htt (c), whereas mutated htt was found mostly in nuclear inclusions (e). The expression of the chaperone proteins was analyzed in hsp104- and hsp27-injected animals (f–i) 12 weeks after injection, using anti-hsp104 and anti-hsp27 antibodies, respectively. Consistent with the in vitro data, hsp104 was detected in the entire cell body (g), whereas hsp27 (i) was present in the cytoplasm.

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Figure 4  Neuroprotective effect of chaperones in a rat model of Huntington’s disease. Analyses of DARPP-32 staining and quantification of the htt171-82Q-induced lesion. (a, b) The expression of htt17182Q in the rat striatum led to a loss of DARPP-32 expression, which was partially rescued in (c, d) hsp104- and (e, f) hsp27-treated animals. (d–f) High-magnification images showing the presence of DARPP-32positive neurons at the center of the zone infected with htt171-82Q in hsp104- and hsp27-treated rats. (g) Quantitative analysis of the striatal lesion (mean ± SEM, **P < 0.01). One-way analysis of variance, F(2, 34) = 20.89, P < 0.001. Post hoc comparison of 82Q with 82Q + hsp104 or 82Q + hsp27, **P < 0.01.

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Figure 5  Changes in the distribution of huntingtin (htt) inclusions upon chaperone gene expression. The expression of hsp104 and, to a lesser extent, hsp27 affects the distribution of htt inclusions. Two antibodies for htt, (a–c) EM48 and (d–f) N-18, and (g–i) one antibody for ubiquitin were used to analyze the formation of htt inclusions. (j–l) Confocal microscopy analysis of 4′,6-diamidino-2-phenylindole (DAPI) and EM48 staining shows the presence of nuclear (arrows; DAPI- and EM48positive staining) and non-nuclear inclusions (arrowheads; EM48-positve and DAPI-negative staining).

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Striatal damage occurs progressively in this model, with an early loss of expression of the neuronal marker DARPP-32, which reflects a dysfunctional state of the neurons, the progressive accumulation of htt inclusions, and cell death over 3 months. Lentiviral vectors encoding hsp104 and hsp27 were first injected into the striatum of adult rats. One month later, vectors expressing wild-type or mutated htt (htt171-19/82Q) were injected at the same coordinates (Figure 3a). The rats were killed 2 months after injection of the htt171-19/82Q vectors and expression of htt was examined with the 2B4 antibody. Diffuse, cytoplasmic staining was observed with the wild-type htt fragment (Figure 3b and c), whereas large numbers of nuclear htt inclusions were observed in htt171-82Q-infected striata (Figure 3d and e). At 12 weeks after injection, the subcellular distribution of the exogenous chaperones was similar to that in primary striatal cell cultures, with accumulation of hsp104 in the entire cell body (Figure 3f and g) and a cytosolic localization of hsp27 (Figure 3h and i). The intracellular localization of hsp104 and hsp27 was not affected by the co-expression of htt171-19Q (Supplementary Figure S1A and C) or htt171-82Q (Supplementary Figure S1B and D). Immunostaining for the GABAergic neuronal marker DARPP32 supported the view that the overexpression of hsp104 or hsp27 (alone or in combination with htt171-19Q) was not toxic to medium spiny neurons in vivo (Supplementary Figure S2A–J). 906

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Figure 6  Number and size of EM48-positive huntingtin (htt) inclusions. (a) Semi-quantitative analysis of the distribution by size/localization of EM48-positive inclusions. Data are mean ± SEM of the chaperone versus the 82Q control for each subgroup of inclusions. One-way analysis of variance (ANOVA) for nuclear inclusions >10 µm2, F(2, 69) = 15, 58. Post hoc comparison of 82Q with 82Q + hsp104 or 82Q + hsp27, ***P < 0.001. One-way ANOVA for non-nuclear inclusions >10 µm2 is not significant, F(2, 69) = 2.19. One-way ANOVA for nuclear inclusions <10 µm2 is not significant, F(2, 69) = 1.39. One-way ANOVA for nonnuclear inclusions <10 µm2, F(2, 69) = 76.56. Post hoc comparison of 82Q with 82Q + hsp104 or 82Q + hsp27, ***P < 0.001. (b, c) A shift in distribution toward small objects corresponding to non-nuclear inclusions was observed when htt171-82Q was expressed with hsp104 or hsp27. Data are mean ± SD. ***P < 0.0001, **P < 0.005, and *P < 0.05 versus htt171-82Q.

In contrast, DARPP-32 expression was lost in a large area corresponding to 16.9 ± 1% of the striatum of htt171-82Qinjected rats (Figure 4a, b and g), and this striatal pathology was significantly reduced in hsp104- (Figure 4c, d and g) and hsp27-treated animals (Figure 4e–g). We carried out immunohistochemical staining with antibodies against htt and ubiquitin to determine whether this protection was accompanied by a change in inclusion formation. The co-expression of htt171-82Q and hsp104 (Figure 5b, e, h and k) and, to a lesser extent, the www.moleculartherapy.org vol. 15 no. 5 may 2007

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Figure 7  Induction of endogenous hsp70 expression by htt171-82Q. The levels of endogenous hsp70 were lower in the control htt171–19Q group (a, b) compared to the increase in endogenous hsp70 expression induced by the expression of mutated htt (c, d). (d) In htt171-82Qexpressing neurons, the hsp70 staining was found mostly in nuclear inclusions. The co-expression of htt171-82Q with hsp104 (e, f) or hsp27 (g, h) altered the subcellular distribution of hsp70, which became more diffuse and cytoplasmic.

co-expression­ of htt171-82Q and hsp27 (Figure 5c, f, i and l) resulted in larger numbers of small htt inclusions than observed with htt171-82Q (Figure 5a, d, g and j). This effect was further analyzed with the quantification of all EM48-positive inclusions (Figure 6b and c). The small neuritic inclusions (0–4 µm2) were found to be 5.4 times more abundant in hsp104-treated animals and 2.5 times more abundant in hsp27-treated animals than in animals expressing mutated htt alone (Figure 6b and c). The number of medium-sized (20–30 µm2) inclusions was significantly reduced in chaperone-treated animals. Semi-quantitative analysis of EM48/4′,6-diamidino-2-phenylindole double-stained sections using confocal microscopy demonstrated that these alterations reflect a decrease in the ratio of nuclear versus non-nuclear inclusions (Figure 6a).

Induction of endogenous hsp expression Molecular chaperones are known to work in concert to exert their various beneficial activities. We have shown that mutated htt expression is associated with an increase in hsp70 immunostaining in primary striatal neurons.23 We therefore investigated the possible up-regulation of endogenous chaperones in vivo. We found that endogenous hsp70 levels were higher Molecular Therapy vol. 15 no. 5 may 2007

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Figure 8  Induction of endogenous small heat-shock protein (shsp) expression by htt171-82Q and hsp104. In comparison with neurons expressing (a, b) the wild-type fragment of htt, the endogenous hsp27 was up-regulated in striatal neurons expressing (c, d) htt171-82Q. The overexpression of (e, f) hsp104 alone or (g, h) together with htt17119Q led to the more marked up-regulation of endogenous hsp27 and strong neuronal and fibrillar staining. (i, j) The expression of hsp104 with htt171-82Q further increases the up-regulation of hsp27 expression, which extended over a wider area of the striatum than htt17182Q-expressing neurons.

in htt171-82Q-injected animals (Figure 7c and d) than in the htt171-19Q group (Figure 7a and b). As observed in vitro, hsp70 staining was mostly detected in the nuclei of striatal neurons.23 Interestingly, in chaperone-treated animals, hsp70 staining was more diffuse and extended to cell bodies and neurites (Figure 7e–h). The expression of hsp104 or hsp27 alone did not induce the activation of endogenous hsp70 (data not shown). Recent data suggest that hsp104 requires not only the hsp70/40 co-chaperone system but also the activation of shsps to promote the refolding of aggregated proteins.16,17 We therefore assessed whether expression of the endogenous hsp27 was up-regulated by administration of exogenous htt or hsp104. The injection of htt171-19Q induced a weak hsp27 expression, mostly in fibrils 907

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close to the needle track (Figure 8a and b). In htt171-82Q-injected animals, hsp27 expression extended over a wider area, with staining detected in both neurites and cell bodies (Figure 8c and d). Interestingly, hsp27 expression was stronger in animals overexpressing hsp104 (Figure 8e–j) than in controls, but the highest levels of hsp27 up-regulation were observed in rats co-expressing htt171-82Q and hsp104 (Figure 8i and j). Thus, endogenous hsp27 is up-regulated by HD and hsp104 expression further increases hsp27 expression.

DISCUSSION These experiments in primary striatal cultures and in a rat model of HD demonstrated that the overexpression of hsp104 and hsp27 reduces polyQ toxicity. The constitutive overexpression of ­ chaperones prevented the loss of the neuronal marker NeuN and cell death at 8 weeks in primary striatal neurons expressing htt171-82Q. In vivo, expression of the GABAergic projection neuron marker DARPP-32 was preserved in chaperone-treated ­animals. This rescue of DARPP-32 expression was accompanied by a change in htt inclusion distribution, with an increase in the number of small non-nuclear inclusions and a reduction of nuclear htt aggregates. A cellular response characterized by the induction of endogenous hsp70 and hsp27 was observed in rats expressing mutated htt. hsp104 and hsp27 overexpression restores the subcellular localization of hsp70 to the cytoplasmic compartment and hsp104 expression up-regulates the expression of endogenous hsp27. The most obvious candidate mechanism for chaperone­mediated rescue of HD neurotoxicity is the inhibition or reversal of htt aggregate (fibrils/polymers) formation. There is substantial evidence that toxic forms of the mutated htt protein exist, despite the debate as to their exact molecular compositions or relative contributions to pathology. Although the known activities of hsp104 and hsp27 associate them directly with this potential anti-aggregation mechanism, the up-regulation of other endogenous chaperones could also contribute synergistically to the effects of hsp104 and hsp27. For example, a recent study demonstrates that shsps facilitate the disaggregating activity of hsp104.16 Following protein disaggregation and conversion of large htt aggregates into smaller ones by hsp104, the inclusions are recognized either by hsp70/40 or the shsp chaperones and are refolded.17 The activation of endogenous hsp70 and hsp27 in hsp104-treated animals may thus be part of the cascade of events leading to the protective effect of this chaperone. Consistent with the idea that chaperones may decrease protein aggregation, previous studies have shown that hsps prevent polyQ toxicity and that such protection in other model ­systems is associated with an effect on the formation of htt inclusions.9,11,12,18–20 Thus, it was somewhat surprising that neither hsp104 nor hsp27 had a major effect on the total number of EM48-positive inclusions in our experiments. In vivo, the co-expression of chaperones did alter the subcellular localization and morphology of htt inclusions, and this may have important ramifications still to be determined (see below). This increase of non-nuclear aggregates was also observed in our in vitro model, but to a lesser extent. This might be due to the presence of a large number of neuritic aggregates in htt171-82Q-expressing striatal neurons that are not 908

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observed in vivo. This may reflect a lower transgene expression in primary cultures than in vivo.23 The overexpression of chaperones in primary striatal neurons might therefore marginally promote the accumulation of mutated htt in the cytoplasmic compartment. Our findings, however, do not exclude the possibility that the overexpression of chaperones in our rodent HD models has decreased the formation of toxic htt aggregates that are not detectable by light microscopy. Moreover, the rather limited effect of hsps on the total number of htt inclusions might be partially explained by the higher survival of GABAergic neurons in hsptreated animals than in the htt171-82Q group and the progressive accumulation of htt aggregates over time despite the refolding of mutated htt by the chaperones. Several studies have shown that polyQ pathologies are influenced by the subcellular localization of the mutated proteins.27,28 In a SCA1 transgenic mouse model, removal of the nuclear localization signal of ataxin-1 resulted in the production of a non-pathogenic, cytosolic protein.28 Conversely, in cell and transgenic mouse models of HD, the insertion of a nuclear localization signal at the N-terminus of htt dramatically exacerbates the ­phenotype,27,29 and the insertion of a nuclear export signal reduces it.30 The accumulation of neuritic htt inclusions in hsp-treated animals may therefore be associated with lower intrinsic polyQ toxicity in the cytoplasm or may favor the clearance of toxic forms of htt.31–33 Autophagy, the degradation of cytosolic components by lysosomes, was recently shown to play an important role in preventing the accumulation of misfolded proteins in the central nervous system.34,35 Moreover, autophagy has been implicated in the elimination of cytoplasmic but not nuclear polyQ inclusions.31,33,36 We have so far been unable to label cytoplasmic htt aggregates with the autophagic marker LC3, however (data not shown).31,33 Interestingly, cytosolic proteins can be specifically targeted for degradation by a chaperone complex that includes hsp70 in a process known as chaperone-mediated autophagy,37 representing a possible mechanism for hsp-mediated neuroprotection. Thus, additional studies will be required to establish whether autophagy or chaperone-mediated autophagy underlies the observed neuroprotective effects of hsp104 and hsp27. Additional pathways may also contribute to the beneficial effect of chaperones. In our experiments, the overexpression of hsp27 alone was protective in vitro and in vivo. In fact, shsps are often up-regulated in neurodegenerative disorders38 and a mutation in the hsp27 gene has been identified in patients with Charcot–Marie–Tooth disease, the most common inherited neuromuscular disease.39 It has been previously demonstrated that hsp27 protects against polyQ-induced oxidative stress without suppressing polyQ inclusion formation.22 In addition, hsp27 interacts with intermediate filaments and maintains the integrity of intermediate filament networks in cells.40,41 Thus, hsp27 might play a role in the maintenance of axonal cytoskeleton and transport, which are altered in HD.42 Although additional studies will be required to determine their precise mechanism(s) of action, the data from our study confirm the important role of chaperones in protein quality control in the central nervous system. These data suggest that we may be able to delay the onset of the pathological features of HD by increasing chaperone expression. www.moleculartherapy.org vol. 15 no. 5 may 2007

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MATERIALS AND METHODS Plasmids. The complementary DNA encoding the yeast hsp104 (Genbank

M67479; nucleotides 334–3,060)43 was inserted downstream from the mouse phosphoglycerate kinase 1 (PGK) promoter in a self-­inactivating lentiviral transfer vector (SIN-W-PGK),44 such that a Flag epitope (DYKDDDDK; Sigma-Aldrich, Buchs, Switzerland) was present to the N-terminus of the protein (SIN-W-PGK-hsp104). A Kozak consensus sequence (CCACC) was inserted upstream from the ATG. The rat hsp27 complementary DNA (Genbank M86389; nucleotides 44–664) was generously provided by Dr. C.J. Woolf, Massachusetts General Hospital and Harvard Medical School, Boston, MA.45 It was inserted into the SIN-WPGK transfer vector (SIN-W-PGK-hsp27). Lentiviral vector production. Lentiviral vectors encoding yeast hsp104, rat

hsp27, and the first 171 amino acids of the human htt protein with 19 (19Q, normal) or 82 (82Q, mutated) CAG repeats26 were produced in 293T cells, concentrated by ultracentrifugation and re-suspended in 1% bovine serum albumin in phosphate-buffered saline (PBS) as previously reported.46 Viral particle content was defined by p24 antigen enzyme-linked immunosorbent assay (RETROtek, Gentaur, Paris, France). Viral stocks were stored at −80 °C until use. Primary cell cultures. E16 rat striatal primary cultures were established

using a previously described procedure.23 In brief, cells were plated, at a density of 150,000 cells per well, in 48-well dishes (COSTAR, Corning, Cambridge, MA) coated with 0.1 mg/ml of poly-l-lysine (molecular weight 30,000–70,000, Sigma, Buchs, Switzerland). On in vitro day 1, the cultures were infected with the htt171-19Q or htt171-82Q lentiviral vector or with a combination of htt171-19Q/82Q and vectors encoding hsp104 or hsp27 (1:1 ratio, 15 ng p24 for each vector). On in vitro day 4, half the medium was replaced with freshly prepared neurobasal medium supplemented with 1% B27 (Gibco, Invitrogen, Basel, Switzerland), 2% penicillin–streptavidin (10,000 U/ml, 10,000 Ag/ml), 0.5 mmol/l l-glutamine, and 15 mmol/l KCl. Subsequently, half the medium was changed weekly. In vivo experiments

Animals. All experiments were carried out in accordance with the European Community directive (86/609/EEC) for the care and use of laboratory animals. Adult female Wistar rats (Iffa Credo/Charles River, Les Oncins, France) were introduced into the experiment at a weight between 180 and 200 g. The animals were housed in a controlledtemperature room maintained on a 12-hour day/night cycle, with food and water provided ad libitum. Lentiviral injections. Concentrated viral stocks were thawed on ice and re-suspended by repeated pipetting. Lentiviral vectors expressing htt17119Q, htt171-82Q, hsp104, and hsp27 were stereotaxically injected into the striata of animals anesthetized with ketamine (75 mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitoneally) using a 34-gauge blunt-tipped needle linked to a Hamilton syringe (Hamilton, Reno, NV) via a polyethylene catheter. The stereotaxic coordinates used for the injection were 0.5 mm rostral to bregma, 3 mm lateral to midline, and 5 mm from the skull surface. The particle content of each concentrated virus was adjusted to 120,000 ng p24 per ml. We injected 4 µl of viral suspension, at a rate of 0.2 µl/min, into the striatum via an automatic injector (Stoelting, Wood Dale, IL). The needle was left in place for 5 minutes after the injection. The skin was closed with wound chip autoclips (Phymep, Paris, France). The vectors encoding the chaperones were injected 1 month before the htt-expressing vectors. As control, eight rats were injected on the left striatum with PBS–BSA instead of htt171-19Q. Histological processing Primary cell culture. Eight weeks after dissection and the establishment of

the cell culture, the cells were fixed by incubation with cold 4% paraformaldehyde (Fluka, Sigma, Buchs, Switzerland) for 10 minutes at 4 °C. The Molecular Therapy vol. 15 no. 5 may 2007

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cells were then stored in PBS at 4 °C until immunohistochemical staining. NeuN (1:500, Chemicon International, Temecula, CA) staining was carried out as follows. Cells were incubated for 1 hour in a blocking solution­ containing 10% normal goat serum (NGS, Gibco, Invitrogen, Basel, Switzerland), 0.1% Triton X-100 (TX, Fluka, Sigma, Buchs, Switzerland) in PBS. They were incubated overnight at 4 °C with the primary antibody in 5% NGS and 0.1% TX in PBS. Cells were rinsed three times with PBS and then incubated for 1 hour with the fluorescent secondary antibody (goat anti-mouse Cy3, 1:1,000, Jackson Immunoresearch Laboratories, West Grove, PA). The same protocol was used for the rabbit anti-EM48 antibody (1:600, kindly provided by Dr. X.J. Li, Emory University School of Medicine, Atlanta, GA47) with a goat anti-rabbit secondary antibody coupled to Oregon-Green (1:500, Molecular Probes, Eugene, OR). Rabbit EM48– stained sections were counterstained with propidium iodide (500 nmol/l, 1–2 minutes, Fluka, Sigma, Buchs, Switzerland) by first pre-treating the cells with 100 µl/ml DNase-free RNase for 20 minutes at 37 °C in 2× SSC (0.3 mol/l NaCl, 0.03 mol/l sodium citrate, pH 7.0). In vivo experiments. Two months after htt lentiviral injection, the animals were killed by sodium pentobarbital overdose and transcardially perfused with phosphate buffer followed by a fixative solution composed of 4% paraformaldehyde, 10% picric acid in phosphate buffer. Brains were removed and post-fixed by incubation in 4% paraformaldehyde, 10% ­picric acid in phosphate buffer for at least 24 hours. They were then cryoprotected by incubation in 25% sucrose, 0.1 M PBS for 48 hours. A sledge microtome with a freezing stage at −20 °C (SM2400, Leica Microsystems AG, Glattbrugg, Switzerland) was used to cut coronal sections (25 µm). These sections were collected and stored in 96-well plates, free-floating in PBS supplemented with 0.12 µmol/l sodium azide. The plates were stored at 4 °C before immunohistochemical processing. Striatal sections from injected rats were processed for the immunohistochemical staining of dopamine and cyclic adenosine monophosp­hate– regulated phosphoprotein with a molecular mass of 32 kd (DARPP-32, 1:8,000, Chemicon International, Temecula, CA), htt (mouse monoclonal 2B4, 1:200, a gift from Y. Trottier, CNRS/INSERMLP, Illkirch, France),48 hsp104 (anti-hsp104, 1:1,000, StressGen, Victoria, Canada), and hsp70 (anti-hsp70, 1:3500, StressGen, Victoria, Canada) as previously described.49 For the immunohistochemical analysis of hsp27, striatal sections were incubated for 2 hours at room temperature with an anti-hsp25 antibody (murine homolog of hsp27, 1:4,000, StressGen, Victoria, Canada). For the immunohistochemical analysis of ubiquitin (1:1,000, Dakocytomation, Zug, Switzerland), sections were incubated in a blocking solution containing 10% NGS and 10% fetal calf serum (Gibco, Invitrogen, Basel, Switzerland). Sections were stained with EM48 (MAB5374, 1:2,000, Chemicon International, Temecula, CA) and htt N-18 antibodies (N-18, 1:1,000, Santa Cruz Biotechnology, CA) as described below. Sections were incubated for 30 min in 1% sodium cyanoborohydride and rinsed twice in 0.4% TX in PBS. They were then incubated overnight at room temperature with the primary antibody in PBS. Sections were washed six times with 0.4% TX in PBS and then with the corresponding secondary antibody for 2 hours at room temperature. Horseradish peroxidase–conjugated ­rabbit anti-goat antibodies (1:200, Dakocytomation, Zug, Switzerland) were used to detect N-18 and biotinylated horse anti-mouse (rat absorbed) antibodies (1:200, Vector Laboratories, CA) were used to detect EM48. Immunoreactivity was visualized using the Vectastain Elite ABC detection kit (Vector Laboratories, West Grove, PA) and 3,3′-diaminobenzidine-tetrahydrochloride (DAB Metal Concentrate, Pierce, IL) according to the manufacturer’s recommendations. The sections were mounted, dehydrated by passing twice through ethanol and toluene, and cover-slipped with Merckoglas (EM Science, Gibbstown, NJ). A secondary fluorescent antibody was used for detection as follows. Sections were first incubated for 1 hour at room temperature in PBS supplemented with 10% NGS and 0.1% TX. The EM48 antibody (1:200) was incubated with samples overnight at 4 °C in PBS supplemented with 5% NGS and 0.1% TX. Sections were then

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processed by incubation with a fluorescent goat anti-mouse Cy3 antibody (for detection of mouse EM48) or Oregon-Green goat anti-rabbit antibody (for detection of rabbit EM48), diluted 1:200, for 1 hour at room temperature. Rabbit EM48–positive sections were counterstained with 4′,6-diamidino-2-phenylindole by incubation in a 1 µg/ml solution for 15 minutes (Fluka, Sigma, Buchs, Switzerland). The sections were mounted and cover-slipped in Mowiol (Calbiochem, Juro, Lucerne, Switzerland). Images and data analysis. For in vitro experiments, we analyzed the

loss of NeuN staining using an Olympus CK40 microscope (­Alberstland, Denmark)­. At ×40 magnification, we counted ten fields of view for each sample. Duplicates of three independent experiments (n = 6) were analyzed for each group. The number of NeuN-positive stained cells for each group was expressed as a mean of the six values obtained ± SEM. For statistical analysis, we carried out one-way analysis of variance, followed by a Newman–Keuls post hoc test (Statistica 5.1; Statsoft, Maisons-Alfort, France). Values of P < 0.05 were considered significant. For in vivo experiments, the loss of DARPP-32 expression was analyzed by digitizing images of 12 sections per animal (spaced by 150 µm) and quantifying optical density with image analysis software (National Institutes of Health Image, Version 1.62, National Institutes of Health, Bethesda, MD). We analyzed sections distributed throughout the striatum. Data are expressed as the ratio of DARPP-32 optical density, corresponding to the optical density measured on the entire striatum from the lesioned (82Q) side minus the background divided by the optical density on the non-lesioned (19Q) side minus the background. The corpus callosum and the anterior commissure were used to delineate the striatal area. The background was defined as the average signal of the entire cortex surrounding the striatum on the same section. n = 9 for the htt (control) group, n = 12 for the hsp104 group, and n = 16 for the hsp27 group. One animal in the htt group and four animals in the hsp104 group were not included in the analysis because injection was not made at the correct site. The level of DARPP-32 signal was expressed as a mean value ± SEM. Statistical analysis was performed by one-way analysis of variance followed by a Newman–Keuls post hoc test (Statistica 5.1, Statsoft). Values of P < 0.05 were considered significant. Analysis of EM48 aggregate distribution

In vitro confocal microscopy analysis. Double-stained EM48/propidium iodide striatal cultures were used to analyze the nuclear/non-nuclear distribution of htt inclusions. Confocal images of striatal cultures were acquired using a Zeiss confocal microscope (LSM510/Axiovert 220M, Thornwood, NY). The analysis of EM48-positive inclusion localization was performed for 16 areas from 4 different striatal cultures for each group. The EM48positive inclusions were then quantified as nuclear or non-nuclear based on the propidium iodide counterstaining. The data are represented as the average of the difference between nuclear and non-nuclear inclusions for each group. Statistical analysis was performed using a two-tailed paired t-test (Microsoft Excel software, Microsoft, Seattle, WA). Values of P < 0.05 were considered significant. In vivo confocal microscopy analysis. EM48 signals in brain sections counterstained with a nuclear marker (4′,6-diamidino-2-phenylindole) were used to analyze the intracellular distribution of htt aggregates (EM48 staining) and to compare that distribution with nuclear staining (4′,6-diamidino-2-phenylindole staining). Images were acquired with a Leica confocal microscope (Confocal Leica TCS-SP2 AOBS, Leica Microsystems AG, Glattbrugg, Switzerland) and analyzed as indicated in Supplementary Materials and Methods.

ACKNOWLEDGMENTS We thank for expert technical assistance Fabienne Pidoux, Vivianne Padrun, Maria de Fatima Rey, Christel Sadeghi, Anne Maillard, and Philippe Colin from the Ecole Polytechnique Fédérale de Lausanne­ (EPFL); and Noelle Dufour, and Gwennaelle Auregan from the ­ Commissariat

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à l’Energie Atomique (CEA). This work was supported by the Swiss ­National Science Foundation, the Commissariat à l’Energie Atomique, and the Ecole Polytechnique Fédérale de Lausanne.

SUPPLEMENTARY MATERIAL Figure S1. Subcellular distribution of the chaperone proteins and of htt. Figure S2. Expression of hsps alone or with htt171-19Q does not lead to DARPP-32 down-regulation. Materials and Methods.

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