Expansion of a stretch of polyglutamine in huntingtin (htt), the protein product of the IT15 gene, causes Huntington's disease (HD). Previous investigations into the role of the polyglutamine stretch (polyQ) in htt function have suggested that its length may modulate a normal htt function involved in regulating energy homeostasis. Here we show that expression of full-length htt lacking its polyglutamine stretch (ΔQ-htt) in a knockin mouse model for HD (Hdh 140Q/ΔQ ), reduces significantly neuropil mutant htt aggregates, ameliorates motor/behavioral deficits, and extends lifespan in comparison to the HD model mice (Hdh 140Q/+ ). The rescue of HD model phenotypes is accompanied by the normalization of lipofuscin levels in the brain and an increase in the steady-state levels of the mammalian autophagy marker microtubule-associate protein 1 light chain 3-II (LC3-II). We also find that ΔQ-htt expression in vitro increases autophagosome synthesis and stimulates the Atg5-dependent clearance of truncated N-terminal htt aggregates. ΔQ-htt's effect on autophagy most likely represents a gain-of-function, as overexpression of full-length wild-type htt in vitro does not increase autophagosome synthesis. Moreover, Hdh ΔQ/ΔQ mice live significantly longer than wild-type mice, suggesting that autophagy upregulation may be beneficial both in diseases caused by toxic intracellular aggregate-prone proteins and also as a lifespan extender in normal mammals.

Expansion of a stretch of glutamines near the amino-terminus of huntingtin (htt), the protein product of the IT15 gene, is a deleterious mutation that causes Huntington's disease (HD). Here we show, in contrast, that deletion of htt's normal polyglutamine stretch (ΔQ-htt) is a potentially beneficial mutation that can ameliorate HD mouse model phenotypes when ΔQ-htt is expressed together with a version of htt with the HD mutation. In addition, ΔQ-htt expression can enhance longevity when expressed in either an HD mouse model or in non–HD mice. ΔQ-htt's effects on both lifespan and HD model phenotypes are likely due to an increase in autophagy, a major recycling pathway in cells that is involved in the turnover of cellular components, and aggregated protein. Based on our results, we suggest that development of therapeutic agents that can stimulate autophagy may help both in treating neurodegenerative disorders like HD and also in increasing longevity.

Funding: This work was funded by NIH NS43466, http://www.nih.gov , to SOZ, the Wellcome Trust Senior Fellowship, http://www.wellcome.ac.uk , to DCR, and the MRC Programme Grant, http://www.mrc.ac.uk/Fundingopportunities/Grants/Programmegrant/index.htm , to DCR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To assess ΔQ-htt function in the presence of expanded polyQ htt expression, we generated mice expressing both ΔQ-htt and 140Q-htt (Hdh 140Q/ΔQ ). We found that ΔQ-htt expression in the HD mouse model rescued behavioral/motor deficits, reduced the number of neuropil htt aggregates, normalized brain lipofuscin levels, and enhanced lifespan relative to the HD mouse model. Clearance of htt aggregates and the accumulation of lipofuscin are mediated by autophagy, a catabolic pathway that encompasses several distinct processes in mammalian cells [17] . Macroautophagy generally involves the non-selective turnover of bulk cytoplasmic contents, including organelles and aggregated protein, and is an essential pathway for the survival of organisms during nutrient deprivation [18] . Upregulation of autophagy reduces truncated mutant htt aggregation and toxicity in both in vitro and in vivo models [19] – [22] , and recently, the acetylation of soluble full-length htt has also been reported to assist its recognition by the autophagic apparatus [23] . In Hdh ΔQ/+ and Hdh 140Q/ΔQ mice, we observed enhanced microtubule-associated protein 1 light chain 3 (LC3, [24] ) immunostaining, and increased levels of the LC3-II autophagic marker. Expression of ΔQ-htt, but not wild-type htt, induced the formation of autophagosomes in SK-N-SH neuroblastoma cells, and enhanced the clearance of truncated 74Q-htt aggregates in an autophagy-dependent process. Based on our observations, we hypothesize that deletion of the polyQ stretch within huntingtin enhances neuronal macroautophagy resulting in the more efficient clearance of neuropil mutant htt and phenotypic rescue in Hdh 140Q/ΔQ mice. Moreover, we have observed that mice homozygous for ΔQ-htt expression live significantly longer than wild-type mice, an observation that is compatible with the view that enhancing constitutive autophagy may also be beneficial in normal ageing.

In lymphoblastoid cell lines derived from HD patients, polyQ length (in both the normal and mutant htt alleles) affects energy status, with a longer polyQ stretch correlating with a reduced cellular ATP/ADP ratio [15] . Deletion of the normal short polyQ stretch (7Q) in mouse htt (ΔQ-htt) also results in elevated ATP levels in fibroblasts derived from embryonic and adult Hdh ΔQ/ΔQ mice [16] . In addition, adult Hdh ΔQ/ΔQ mice exhibit subtly enhanced performance on the rotarod, and altered behavior in the Barnes maze learning and memory test.

In vertebrates, the polyQ stretch within htt is located close to the protein's N-terminus, and separates a highly conserved 17 amino acid N-terminal domain (N1–17) that can act as a membrane association signal [1] , from a proline-rich region that is implicated in protein-protein interactions [2] – [4] . Expansion of htt's polyQ stretch (>37Q) causes Huntington's disease (HD), a neurodegenerative disorder characterized by the appearance of cytoplasmic (neuropil) and nuclear aggregates of mutant htt, and selective cell death in the striatum and cortex [5] – [9] . Although the mechanism of pathogenesis is still unclear, HD is recognized as a toxic gain-of-function disease, where the expansion of the polyQ stretch within htt confers new deleterious functions on the protein. The extent to which the polyQ expansion affects normal htt function is also unclear, although there is accumulating evidence that loss of normal htt function likely contributes to HD pathogenesis [10] . The polyQ stretch is conserved in vertebrate htt, and its non-pathogenic size varies from 4Q in fish, to 37Q in humans [11] – [13] . However, the polyQ stretch is absent in Ciona and Drosophila htt, and present as only a short hydrophilic NHQQ stretch in sea urchin htt, suggesting that addition of a htt polyQ stretch may be a late evolutionary feature acquired sometime after protostome-deuterostome divergence [14] .

Results

Rescue of Hdh140Q/+ motor and behavioral deficits in Hdh140Q/ΔQ mice To evaluate the impact of expressing a version of wild-type htt lacking its short polyQ stretch on the motor and behavioral phenotypes exhibited by a mouse model for HD, HdhΔQ/+ mice were crossed with the CAG140 knock-in mouse expressing full-length htt with a chimeric human/mouse htt exon 1 containing an expanded stretch of 140 glutamines [25], (for a diagram of the knockin alleles used in this study, see Figure 1A). Hdh140Q/ΔQ, Hdh140Q/+, and wild-type control littermates were assessed using the accelerating rotarod, the Barnes maze, and an activity cage. Mice were tested on an accelerating rotating rod at 1, 5, and 19 months of age (Figure 1B). At one month of age, there were no significant differences between the wild-type controls, Hdh140Q/+ mice, and the Hdh140Q/ΔQ mice (n = 6 for each genotype at 1 and 5 months, n = 4 of each genotype at 19 months). A two-way repeated measures ANOVA showed no significant effect of genotype (F (2,6) = 0.87; P>0.05), although there was a significant trial day effect (F (4,6) = 13.00; P<0.001), indicating that all mice were learning to stay on the rod. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. ΔQ-htt expression ameliorates motor and behavioral deficits in Hdh140Q/ΔQ mice. (A) Diagram of the Hdh140Q, wild-type (Hdh7Q), and HdhΔQ exon 1, and a schematic of the breeding scheme used to generate the mice employed in this study. The expansion of the polyQ stretch in the Hdh140Q allele also includes human exon 1 sequences (purple) between a conserved XmnI restriction site within exon 1 and a KpnI restriction site located within intron 1, while the deletion of the polyQ stretch in the HdhΔQ allele also includes the insertion of a FLAG epitope tag (green) following the initiation codon. Endogenous mouse sequence is shown in light blue. (B) Accelerated rotarod testing of wild-type (+/+), Hdh140Q/+ (140Q/+), and Hdh140Q/ΔQ (140Q/ΔQ) mice (n = 6 for each genotype). Hdh140Q/+ versus +/+ at 5 months, ANOVA for genotype; P<0.03 and at 19 months; P<0.04. The performance of the Hdh140Q/ΔQ mice did not differ significantly from the wild-type controls. (C) Barnes maze testing of 5 month old wild-type (+/+), Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 5 of each genotype). Hdh140Q/+ mice performed poorly on their distance scores compared to wild-type and Hdh140Q/ΔQ mice (ANOVA for genotype; F (2,4) = 5.96, P<0.02). Hdh140Q/ΔQ and wild type mice also made significantly fewer errors than Hdh140Q/+ mice before finding the target (ANOVA for genotype; F (2,4) = 25.28; P<0.001). *Significant differences on individual trial days between Hdh140Q/ΔQ and Hdh140Q/+ mice (Holm-Sidak post hoc analysis; P<0.001 to 0.05). (D) Horizontal activity in a novel environment was assessed in wild-type, Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 5 of each genotype) at 6 and 20 months of age. At both ages, the Hdh140Q/+ mice were significantly more hypoactive than the Hdh140Q/ΔQ and wild- type mice (one way ANOVA F(2,12) = 6.63, P<0.02 and Bonferroni post-hoc analysis, P<0.02 at 6 months; one-way ANOVA (F(2,14) = 6.78, P<0.02 and Bonferroni post-hoc analysis Hdh140Q/ΔQ versus Hdh140Q/+ at 20 months, P<0.01). https://doi.org/10.1371/journal.pgen.1000838.g001 At five months of age, however, the Hdh140Q/+ mice performed poorly in comparison to both the wild-type control group and the Hdh140Q/ΔQ group (genotype effect; F (2,6) = 5.4; P<0.03). Interestingly, at five months, the Hdh140Q/ΔQ mice were indistinguishable from the wild-type controls. At 19 months of age, both the wild type and Hdh140Q/ΔQ mice still performed better than the Hdh140Q/+ mice and were indistinguishable from each other (genotype effect; F (2,4) = 6.5; P<0.04), although all mice were performing more poorly at 19 months relative to their performance at 5 months of age. At five months of age, the mice were also tested on the Barnes maze, a measure of spatial learning and memory [26]. Wild-type mice produced better scores on the Barnes maze distance test than Hdh140Q/+ mice, but did not differ significantly from the Hdh140Q/ΔQ mice (n = 5 of each genotype) (Figure 1C). The distance score measures how effectively the mice are using spatial cues to locate the escape tunnel. A two way repeated measures ANOVA revealed a significant effect of genotype (F (2,4) = 5.96; P<0.02) and a significant effect of trial day (F (8,4) = 2.2; P<0.04). In addition, wild-type and Hdh140Q/ΔQ mice made fewer errors than Hdh140Q/+ mice before finding the Barnes maze target (Figure 1C). A two-way repeated measures ANOVA revealed a significant effect of genotype (F (2,4) = 25.28; P<0.001), and a significant effect of trial day [(F (8,4) = 3.33; P<0.003)]. At 6 and 20 months of age, the mice were also tested in an activity cage (n = 5 of each genotype) (Figure 1D). Previous analyses of Hdh140Q mice revealed that they exhibit a period of hyperactivity, followed by hypoactivity when tested at night in an activity cage [25]. Based on total horizontal activity, the Hdh140Q/+ mice were more hypoactive at night than the wild-type mice at 6 months, but the exploratory activity of the Hdh140Q/ΔQ mice did not differ significantly from wild-type controls (one-way ANOVA F (2,12) = 6.63; P<0.02; post-hoc analysis wild-type versus Hdh140Q/+, P<0.02). At 20 months of age, one-way ANOVA revealed an overall difference in activity levels as well (F (2,14) = 6.78; P<0.02). Bonferroni post-hoc analysis showed the Hdh140Q/+ mice to be significantly hypoactive when compared to the Hdh140Q/ΔQ mice (P<0.01). Hdh140Q/ΔQ mice also exhibited a significant increase in their lifespan (median age of 31+/−0.8 months) in comparison to either Hdh140Q/+ or Hdh140Q/140Q mice (median ages of 24+/−2.3 and 27+/−1.7 months, respectively, Hdh140Q/+ versus Hdh140Q/ΔQ log-rank test, χ2 = 11.7, P<0.002; Hdh140Q/140Q versus Hdh140Q/ΔQ log-rank test, χ2 = 9.9, P<0.003 for n = 8 females of each genotype) (Figure 2). However, we could not detect any significant difference in the lifespan of the Hdh140Q/+ and Hdh140Q/140Q mice (log-rank test, χ2 = 0.03, P = 0.958). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. HdhΔQ expression enhances longevity in a knockin mouse model for HD. Kaplan-Meier survival curves are shown for Hdh140Q/+, Hdh140Q/140Q, and Hdh140Q/ΔQ mice (n = 8 mice of each genotype). The Hdh140Q/ΔQ mice lived significantly longer than either the Hdh140Q/140Q mice or the Hdh140Q/+ mice (log-rank test; χ2 = 9.9, P<0.003 and χ2 = 11.7, P<0.002, respectively). The lifespans of the Hdh140Q/+ and Hdh140Q/140Q mice did not differ significantly (χ2 = 0.03, P = 0.958). https://doi.org/10.1371/journal.pgen.1000838.g002

Reduction of neuropil htt aggregates in Hdh140Q/ΔQ mice To determine if the rescue of behavioral phenotypes in the Hdh140Q/ΔQ mice correlated with a change in the number and distribution of htt aggregates, we examined Hdh140Q/+, Hdh140Q/ΔQ, and HdhΔQ/+ (control) brains (n = 4 of each genotype) using an antibody recognizing aggregated mutant htt in inclusions (MW8 [27]) (Figure 3A). At 4 months of age, we were unable to detect htt aggregates in either Hdh140Q/+ or Hdhı4˜Q˜ΔQ mice. Starting at 6 months of age, however, we observed a small, but similar number of nuclear aggregates in the striatum of both genotypes. In contrast, there was a significant reduction in the number of striatal neuropil aggregates observed at 6 months of age in the Hdh140Q/ΔQ brain in comparison to the Hdh140Q/+ brain, P<0.001 (Figure 3B). At 1 year and 2 years of age, the aggregate load increases dramatically in the Hdh140Q/+ brain, with the number of striatal neuropil aggregates growing more quickly with age than the number of nuclear aggregates (Figure 3B). The significant reduction in the number of striatal neuropil aggregates that was observed at 6 months of age in the Hdh140Q/ΔQ striatum was also observed in the striatum of Hdh140Q/ΔQ mice at 1 year and 2 years of age, P<0.001 and P<0.05, respectively. In the cortex, a similar marked decrease in Hdh140Q/ΔQ neuropil aggregates was observed at 6 months, 1 year, and 2 years of age (P<0.001–P<0.005) (Figure S1). In both striatum and cortex, nuclear aggregates were also reduced significantly at 1 year of age, but the magnitude of the decrease was less than that observed for the neuropil aggregates. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Reduced htt neuropil aggregates in the Hdh140Q/ΔQ striatum. (A) Representative confocal images of the striatum from HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice at 4 months, 6 months, 1 year, and 2 years of age that were immunostained with an antibody recognizing htt aggregates (MW8, red signal). Nuclei were stained with To-Pro-3 (blue). Enlarged images of the areas enclosed by dashed white boxes are shown in the bottom panels. Open and solid white arrowheads indicate neuropil and nuclear aggregates, respectively. Scale bars = 25 µm (top panels), 10 µm (bottom three panels). (B) Total; T, nuclear; N, and neuropil; C, htt aggregate numbers from the Hdh140Q/+ and Hdh140Q/ΔQ striatum (n = 4 mice of each genotype). The aggregate numbers (mean ± s.e.m.) represent counts/field from 8 images of the ventral and lateral striatum from each mouse. *P<0.05, **P<0.001. https://doi.org/10.1371/journal.pgen.1000838.g003

Lipofuscin deposits are reduced in Hdh140Q/ΔQ mice Increased lipofuscin has been observed in the HD brain and in the R6/2 transgenic mouse model for HD [28]–[30]. Accumulating in the lysosomes of neurons and other post-mitotic cells, lipofuscin is a yellowish-brown autofluorescent aging pigment that is composed of oxidized lipid and aldehyde cross-linked protein [31]. Lipofuscin is believed to be the byproduct of the incomplete autophagic catabolism of cellular organelles, such as mitochondria that are rich in iron. Iron and peroxide-catalyzed oxidation of incompletely digested lipid and protein results in the slow accumulation of lipofuscin in autolysosomes at a rate that correlates with metabolic activity and age of the organism [32]. In HD, oxidative stress may enhance the formation of lipofuscin, resulting in the appearance of large perinuclear lipofuscin deposits in neurons. In aged cells with high levels of lipofuscin, autophagy is diminished [33],[34], and in C. elegans, lower levels of lipofuscin in age-matched worms correlated with greater motility, suggesting that lipofuscin accumulation reflects biological versus chronological age [35]. We compared the extent of lipofuscin accumulation in the striatum and cortex of wild-type, HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice at 4 months, 6 months, 1 year, and 2 years of age (n = 4 mice of each genotype) (Figure 4A and 4B). Consistent with prior observations in the R6/2 HD transgenic mouse model and in postmortem HD brain tissue, we observed a significant increase in lipofuscin (measured as the pixel area of deposits in confocal images) in the striatum and cortex of Hdh140Q/+ mice as they aged in comparison to wild-type mice, P<0.05 to P<0.001 (Figure 4B). Lipofuscin accumulation was greater in the striatum, relative to the cortex in the Hdh140Q/+ brain. In both the Hdh140Q/ΔQ cortex and striatum, however, neuronal lipofuscin accumulation was similar to that observed in wild type controls at all ages examined. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. ΔQ-htt expression in Hdh140Q/ΔQ mice normalizes the increased lipofuscin accumulation that is observed in the Hdh140Q/+ brain. (A) Confocal images of the parietal cortex and striatum from one year old HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 4 of each genotype). Lipofuscin deposits are yellow (white arrowheads), and nuclei are stained with To-Pro-3 (blue). Scale bar = 25 µm. (B) Lipofuscin deposits at 4 months, 6 months, 1 year, and 2 years of age in wild-type (+/+), HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ parietal cortex and striatum were quantified by measuring their pixel area/field (mean ± s.e.m.). *P<0.05, **P<0.001. https://doi.org/10.1371/journal.pgen.1000838.g004

Altered autophagy in HdhΔQ/+ and Hdh140Q/ΔQ mice To determine if clearance of the neuropil htt aggregates and the reduction in lipofuscin in the Hdh140Q/ΔQ brain may be related to altered autophagy, we performed immunohistochemical analyses and western blot analyses of cellular fractions obtained from wild-type, HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ whole brains and dissected brain regions, respectively, using an antibody to LC3. LC3 is encoded by the mammalian homolog of the yeast Atg8 gene, and is widely used as a marker for autophagy in mammalian cells because it associates tightly with autophagic membranes beginning at vesicle nucleation, and ending with its turnover in autolysosomes [24]. Western blotting with antibodies recognizing the N-terminus of LC3 detects two species with apparent molecular weights of 18 kD (LC3-I) and 16 kD (LC3-II). LC3 is processed proteolytically at its C terminus to form cytosolic LC3-I, which is conjugated to phosphatidylethanolamine on autophagosome membranes to form LC3-II. LC3-II associates specifically with autophagosome and autolysosome membranes, and LC3 vesicle numbers or levels of LC3-II correlate with autophagosome numbers [24],[36]. LC3 immunostaining was enhanced in the striatum of Hdh140Q/ΔQ mice beginning at 6 months of age in comparison to age-matched wild-type, HdhΔQ/+, and Hdh140Q/+ mice (n = 4 of each genotype, Figure S2). At 1 year of age, the Hdh140Q/ΔQ striatum continued to exhibit enhanced LC3 immunostaining, and at 2 years of age, elevated LC3 immunostaining was now detected in both the HdhΔQ/+ and Hdh140Q/ΔQ striatum (Figure 5A). In contrast, LC3 immunostaining in the Hdh140Q/+ striatum at 1 year and 2 years of age was not increased substantially in comparison to age-matched wild-type controls. Moreover, co-localization of LC3 immunostaining with neuropil htt aggregates was observed in the Hdh140Q/ΔQ striatum at 1 and 2 years of age, but was difficult to detect in the Hdh140Q/+ striatum (Figure 5A). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. ΔQ-htt expression in mice enhances LC3 immunostaining and LC3-II steady-state levels. (A) Confocal images of LC3 (green) and htt aggregate (MW8, red) immunostaining in the striatum from 1 year and 2 year old wild-type (+/+), HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 4 of each genotype). Nuclei were stained with To-Pro-3 (blue). Enlarged images of the areas enclosed by dashed white boxes are shown in the bottom panels. White arrowheads indicate examples of htt aggregates co-immunostaining with LC3 (yellow signal). Scale bars = 25 µm (top panels), 10 µm (bottom panels). (B) Western analyses for LC3-I and LC3-II in striatal supernatant (Sup) and pellet (Pel) factions from 2 year old wild-type (+/+), HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 4 of each genotype). Blots were stripped and re-probed with an antibody against β-actin to control for protein loading. The positions of protein standards (in kD) are indicated on the left. (C) Quantification of LC3-II levels in the supernatant and pellet fractions relative to actin. *P<0.004 versus +/+. https://doi.org/10.1371/journal.pgen.1000838.g005 To confirm that the enhanced LC3 immunostaining observed in the HdhΔQ/+ and Hdh140Q/ΔQ striatum was due to an increase in LC3-II levels, dissected striata from 2 year old wild-type, HdhΔQ/+, Hdh140Q/+, and Hdh140Q/ΔQ mice (n = 4 of each genotype) were homogenized and separated into supernatant (NP40-soluble) and pellet (NP40-insoluble) fractions, and then analyzed by western blotting with an antibody that recognizes both LC3-I and LC3-II (Figure 5B and 5C). In the soluble protein fractions, an increase in LC3-II was observed in the Hdh140Q/ΔQ striatum. Interestingly, LC3-II was also enriched in the striatal pellet fractions from both HdhΔQ/+ and Hdh140Q/ΔQ mice. In contrast, LC3-II was present at only low levels in the wild-type and Hdh140Q/+ pellet fractions. A corresponding western blot analysis of LC3 levels in total (unfractionated ) protein extracts from 2 year old mice revealed an increase in LC3-II in both the HdhΔQ/+ and Hdh140Q/ΔQ samples (Figure S3B). We note that we also observed an enrichment of both the autophagy protein beclin 1 and lysosome-associated membrane protein type 1 (Lamp1) levels in the 800×g low-speed P1 fractions from HdhΔQ/+ and Hdh140Q/ΔQ striatal extracts prepared by lysis in the absence of detergent (Figure S3A). Overall levels of beclin 1 and Lamp1 in total brain extract, however, were similar in all genotypes (Figure S3B). Lamp1 is a marker for late endososomes, amphisomomes (formed after autophgagosome-late-endosome fusion), dense autolysosomes and lysosomes that are enriched in the 800×g P1 fraction [37], and these observations, together with our findings related to the alterations in beclin 1 and LC3-II fractionation, suggest that the subcellular distribution of several components of the autophagy pathway are altered by ΔQ-htt expression. It was proposed recently, that htt's association with the ER via its N1–17 domain allows it function as a sensor of ER stress, and to potentially regulate autophagy [1],[38]. In previous work, we found no obvious difference in the nuclear/cytoplasmic localization of ΔQ-htt in comparison to wild-type htt in early passage wild-type and HdhΔQ/ΔQ primary mouse embryonic fibroblasts (PMEFs) [16]. To analyze further the subcellular localization of wild-type- and ΔQ-htt together with markers for the ER (calnexin), and to assess a marker for autophagy (LC3), we performed immunocytochemistry on passage 5 (P5) cultures of wild-type and HdhΔQ/ΔQ primary mouse embryonic fibroblasts (PMEFs) (Figure S4). P5 cultures of wild-type fibroblasts are actively dividing, while P5 cultures of HdhΔQ/ΔQ fibroblasts are, in contrast, undergoing replicative senescence [16]. Wild-type- and ΔQ-htt were detected in both the cytoplasm and nucleus, and perinuclear localization of wild-type- and ΔQ-htt with the ER marker, calnexin was also detected in both Hdh+/+ and HdhΔQ/ΔQ PMEFs (Figure S4A, S4B). However, nuclear localization of htt appeared to be increased in those cells with a more senescent morphology (i.e. more flattened/spread appearance). Perinuclear LC3 immunoreactivity was also enhanced in the HdhΔQ/ΔQ PMEFs with a senescent morphology (Figure S4C), suggesting the possibility for increased autophagy in those HdhΔQ/ΔQ PMEFs undergoing replicative senescence.

Expression of ΔQ-htt enhances autophagosome synthesis in vitro An alteration to autophagy resulting in the increased steady-state levels of LC3-II can be attributed to either enhanced autophagic flux, or to a block in a later step within the pathway that would interfere with the turnover of LC3-II in the autolysosome [39]. To determine if ΔQ-htt can enhance autophagosome synthesis, we transfected SK-N-SH neuroblastoma cells with full-length wild-type (7Q-htt) or ΔQ-htt cDNA expression constructs (diagrams in Figure S5), and monitored the levels of LC3-II 24 h post-transfection by western blotting (Figure 6A). The levels of LC3-II were increased significantly in the ΔQ-htt transfected cells in comparison to either control vector- or 7Q-htt-transfected cells. To monitor autophagy by an alternative method, we also transfected HeLa cells with an EGFP-LC3 expression construct, together with pCDNA3.1 (vector control), 7Q-htt or ΔQ-htt in a 1∶3 ratio (Figure S6). The proportion of EGFP-positive cells with >10 EGFP-LC3 vesicles was assessed and expressed as an odds ratio with 95% confidence limits. ΔQ-htt transfection, but not 7Q-htt transfection, increased the proportion of cells with EGFP-LC3 vesicles. To measure autophagosome synthesis, the cDNA constructs were also transfected in the presence or absence of the antibiotic bafilomycin A 1 , a vacuolar H+ ATPase inhibitor that suppresses turnover of LC3-II in autolysosomes [40]–[42]. Thus, measuring the levels of LC3-II in the presence of bafilomycin A 1 measures LC3-II formation, as the antibiotic blocks LC3-II degradation. The levels of LC3-II were increased significantly in the bafilomycin A 1 -treated and ΔQ-htt-transfected cells in comparison to the bafilomycin A 1 - treated cells alone or the bafilomycin A1-treated and 7Q-htt-transfected cells, suggesting that ΔQ- but not 7Q-htt expression results in increased autophagosome synthesis (Figure 6B). To confirm that an increase in LC3-II formation resulting from ΔQ-htt expression is enhancing autophagic activity that can remove another autophagy substrate, 7Q- or ΔQ-htt constructs were transfected into Atg5+/+ (autophagy-competent) and Atg5−/− (autophagy-deficient) mouse embryonic fibroblasts [43], together with an EGFP-tagged 74Q-htt exon 1 construct (EGFP-HDQ74) expressing an N-terminal fragment of mutant htt that forms aggregates readily in vitro [44]. Aggregate formation in EGFP-positive cells 48 h post-transfection was assessed by calculating odds ratios with 95% confidence limits [44]–[46] (Figure 6C). The proportion of cells with EGFP-HDQ74 aggregates was significantly reduced in Atg5+/+ cells transfected with ΔQ-htt, but not in Atg5−/− cells. Interestingly, 7Q-htt overexpression also reduced aggregate load in both Atg5+/+ and in Atg5−/− cells. These data suggest that while ΔQ-htt can induce autophagic clearance of mutant htt aggregates, 7Q-htt overexpression may induce a reduction in aggregate numbers or formation via an autophagy-independent mechanism in our in vitro system. Taken altogether, these data support the hypothesis that ΔQ-htt expression can stimulate autophagosome formation and the Atg5-dependent clearance of htt aggregates. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. ΔQ-htt expression increases autophagosome synthesis and Atg5-dependent reduction of EGFP-74Q truncated htt aggregates in vitro. (A) SK-N-SH cells were transfected with control vector (pCDNA3.1), 7Q-htt, or ΔQ-htt constructs (transfections performed in triplicate at least twice), and cell lysates were analyzed for LC3-II levels 24 hr post-transfection by western blotting with an anti-LC3 antibody (for comparison, both longer and shorter exposures are shown), and densitometric analysis relative to actin levels. ΔQ-htt versus control; (***P<0.001), ΔQ-htt versus 7Q-htt; (P<0.002). (B) SK-N-SH cells were transfected with control vector, 7Q-htt, or ΔQ-htt constructs and treated with either DMSO (pCDNA3.1 transfected cells) or with 400 nM bafilomycin A 1 (Baf) in DMSO (pCDNA3.1, 7Q-htt, or ΔQ-htt transfected cells) for 4 hours at 20 hr post-transfection. Autophagosome synthesis was analyzed by western blotting using an anti-LC3 antibody and densitometric analysis relative to actin. ***P<0.0001, **P<0.004. (C) Atg5+/+ (autophagy-competent) and Atg5−/− (autophagy-deficient) mouse embryonic fibroblasts were transfected with an EGFP-HDQ74 construct together with vector control, 7Q-htt, or ΔQ-htt constructs, and then assessed for the proportion of EGFP-positive cells with EGFP-74Q-htt aggregates by calculating the odds ratio 48 h post-transfection. ***P<0.0001 for both ΔQ-htt versus control, and for 7Q-htt versus control in Atg5+/+ cells, P<0.0001 for 7Q-htt versus control in Atg5−/− cells. ΔQ-htt's ability to reduce aggregates is abolished in Atg5−/− cells (P = 0.36 in comparison with control vector transfected cells). (D) Hdhex4/5/ex4/5 knock-out (Hdh−/−) mouse embryonic stem (ES) cells were transfected with EGFP along with either pCI (empty vector) or full-length wild-type huntingtin (1∶5 ratio) for 4 h, and then treated with DMSO (– Baf ) or 400 nM bafilomycin A 1 in DMSO (+ Baf) for the last 2 h of the 48 h post-transfection period. Cells were then FACS-sorted for the EGFP-positive cells, in which LC3-II levels were assessed by western blotting and densitometric analysis relative to actin. Htt levels were assessed by western blotting using MAB2166. There were no significant changes in LC3-II levels in the huntingtin transfected Hdh−/− ES cells compared to empty vector transfected Hdh−/− ES cells in the absence (P = 0.7103) or presence (P = 0.4063) of bafilomycin A 1 . https://doi.org/10.1371/journal.pgen.1000838.g006 Importantly, we saw no difference in autophagy in 7Q-htt-overexpressing cells versus empty vector transfected cells (Figure 6A and 6B), or when comparing huntingtin knockout (Hdhex4/5/Hdhex4/5 [47]) mouse embryonic stem cells (Hdh−/−) which were either transfected with empty vector of with wild-type full-length 17Q-Htt (Figure 6D), suggesting that the ability of htt to induce autophagy is a specific consequence of the loss of its polyQ tract.

ΔQ-htt expression stimulates autophagy via an mTOR–independent pathway A central regulator of metabolism and autophagy in both invertebrates and vertebrates is TOR (Target of Rapamycin) kinase, and inhibition of TOR kinase activity by rapamycin and its analogs has been used successfully to stimulate autophagic clearance of mutant htt aggregates in both Drosophila and mouse models for HD [48]. To determine if the activity of mammalian TOR (mTOR) is inhibited by ΔQ-htt expression, we examined the phosphorylation status of mTOR in the striatum of two year old wild-type, HdhΔQ/+, Hdh140Q/+, Hdh140Q/140Q, and Hdh140Q/ΔQ mice, and also the phosphorylation status of downstream targets of mTOR in our in vitro system (Figure 7). Phospho-mTOR (p-mTOR) levels correlate positively with mTOR kinase activity and inversely with mTOR inhibition and the activation of macroautophagy [48], although autophagy can also be regulated by mTOR-independent pathways. We observed no difference in p-mTOR levels in the supernatant fractions of all genotypes examined (Figure 7A). However, we did detect an enrichment of p-mTOR in the striatal pellet fractions from the Hdh140Q/+ and Hdh140Q/140Q brains. This association of p-mTOR with the pellet fraction likely represents p-mTOR association with htt aggregates, as was observed previously both in vitro, and in a transgenic HD mouse model [48]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. ΔQ-htt expression stimulates autophagy through an mTOR–independent mechanism. (A) Western blot analysis of the supernatant (Sup) and pellet (Pel) fractions from the striatum of 2 year old wild-type (+/+), HdhΔQ/+, Hdh140Q/+, Hdh140Q/ΔQ, and Hdh140Q/140Q mice probed with an antibody against phospho-mTOR (p-mTOR). The blots were stripped and re-probed with an antibody (mTOR) recognizing both phosphorylated (enzymatically active form) and non-phosphorylated mTOR (enzymatically inactive form correlating with the induction of autophagy). For protein loading control, a strip from the blot was stained with Ponceau S. The size of standards (in kD) is indicated on the left. (B) SK-N-SH cells transfected with vector control (pCDNA3.1), 7Q-htt, or ΔQ-htt constructs (transfections were performed in triplicate at least twice) were analyzed for mTOR activity 24 h post-transfection by western blotting using antibodies specific for two targets of mTOR kinase activity: S6 kinase (S6K) and S6 ribosomal protein (S6P). The relative levels of phospho-S6K (P-S6K) and phospho-S6P (P-S6P) to total S6K and S6P, respectively, were determined by densitometric scanning of western blots probed with antibodies specific for phospho- and total S6K or S6P. ΔQ-htt expression had no significant effect on the level of P-S6K (7Q-htt versus vector control, P = 0.96; ΔQ-htt versus vector control, P = 0.30) or P-S6P (7Q-htt versus vector control, P = 0.26; ΔQ-htt versus vector control, P = 0.47). https://doi.org/10.1371/journal.pgen.1000838.g007 To confirm our in vivo analyses, SK-N-SH cells were transfected with either 7Q- or ΔQ-htt expression constructs and the phosphorylation status of two targets of mTOR kinase activity were assessed 24 h post-transfection (Figure 7B). The levels of phospho-S6 kinase and phospho-S6 ribosomal protein were not significantly different in the cells transfected with 7Q- or ΔQ-htt, supporting the hypothesis that ΔQ-htt's upregulation of autophagy is not mediated by a reduction in mTOR kinase activity.