There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression. 相似文献
To investigate the in vivo functions of normal prion protein (PrP) in Drosophila, we utilized characterized transgenic flies expressing 3F4-tagged mouse PrP (Mo-PrP3F4). The neurotoxicity of pathogenic Machado-Joseph Disease (MJD) glutamine (Q) 78 and 127Q proteins were enhanced by the co-expression of Mo-PrP3F4 in the fly eyes, while the eyes of controls flies and flies expressing Mo-PrP3F4 alone or together with MJD-Q27 or 20Q proteins did not show any defect. Susceptibilities to H2O2, paraquat, and Dithiothreitol (DTT) were altered in Mo-PrP3F4 flies. In addition, Mo-PrP3F4 flies were significantly more susceptible to the perturbation of autophagy signaling by an autophagy inhibitor, 3-methyladenine (3-MA), and inducer, LiCl. Taken together, our data suggest that Mo-PrP3F4 may enhance the neurotoxicity of pathogenic Poly-Q proteins by perturbing oxidative and autophagy signaling. 相似文献
Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. The molecular mechanisms underlying the selective neuronal death typical of MJD/SCA3 are unknown. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were assayed for the dynamic expression of Hsp27, known as a suppressor of poly-Q mediated cell death, in the presence of mutant ataxin-3 in different passages of cultured cells. A dramatic decrease of Hsp27 expression was observed in the earlier passage of cultured SK-N-SH-MJD78 cells, however, the later passage of cells showed a significant increase of Hsp27 to almost the same level of the parental cells. Furthermore, immunohistochemical analysis of MJD transgenic mice and post-mortem human brain tissues showed increased expression of Hsp27 compared to normal control brain, suggesting an up-regulation of Hsp27 in the end stage of MJD. However, mutant cells of earlier passages were more susceptible to serum deprivation than mutant cells of later passages, indicating weak tolerance toward stress in cells with reduced Hsp27. While heat shock was used to assess the stress response, cells expressing mutant ataxin-3 displayed normal response upon heat shock stimuli when compared to the parental cells. Taken together, we proposed that during the early disease stage, the reduction of Hsp27 synthesis mitigated the ability of neuron cells to cope with cytotoxicity induced by mutant ataxin-3, triggering the cell death process during the disease progress. In the late stage of disease, after prolonged stressful conditions of polyglutamine cytotoxicity, the increased level of Hsp27 may reflect a dynamic process of the survived cells to unfold and remove mutant ataxin-3. However, this increased Hsp27 still cannot reverse the global dysfunction of cellular proteins due to accumulation of cytotoxic effects. 相似文献
Spinocerebellar ataxia type-3 or Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disease caused by triplet nucleotide expansion. The expansion of the polyglutamine tract near the C terminus of the MJD1 gene product, ataxin-3, above a threshold of 40 glutamine repeats causes neuronal loss and degeneration. The expanded ataxin-3 forms aggregates, and nuclear inclusions, within neurons, possibly due to the misfolding of mutant proteins. Here we report upon the behavioral test changes related to truncated and expanded forms of MJD protein (MJDtr) in Drosophila, and show that expanded MJDtr, when expressed in the nervous system, causes characteristic locomotor dysfunction and anosmia. This phenomenon has not been previously reported in humans or in transgenic Drosophila models. In addition, the in vivo expression of the antiapoptotic gene bcl-2 showed no evidence of ameliorating the deleterious effect of MJDtr-Q78s, either in the eye or in the nervous system. The study shows that such Drosophila transgenic models express olfactory dysfunction and ataxic behavior as observed in human patients. 相似文献
Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease‐specific proteins. In case of SCA3, mutation of Ataxin‐3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin‐dependent kinase‐5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase‐dependent Ataxin‐3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease‐propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin‐3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin‐3 and other polyglutamine proteins.
Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known.
Methods
The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured.
Results
We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi.
Conclusions
We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila. 相似文献
Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity. 相似文献
