Nobuhiro Fujikake

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a β-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into β-sheet–rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble β-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic β-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.

Heat shock transcription factor 1-activating compounds suppress polyglutamine-induced neurodegeneration through induction of multiple molecular chaperones.

Many neurodegenerative diseases including Alzheimer, Parkinson, and polyglutamine (polyQ) diseases are thought to be caused by protein misfolding. The polyQ diseases, including Huntington disease and spinocerebellar ataxias (SCAs), are caused by abnormal expansions of the polyQ stretch in disease-causing proteins, which trigger misfolding of these proteins, resulting in their deposition as inclusion bodies in affected neurons. Although genetic expression of molecular chaperones has been shown to suppress polyQ protein misfolding and neurodegeneration, toward developing a therapy, it is ideal to induce endogenous molecular chaperones by chemical administration. In this study, we assessed the therapeutic effects of heat shock transcription factor 1 (HSF1)-activating compounds, which induce multiple molecular chaperones, on polyQ-induced neurodegeneration in vivo. We found that oral administration of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) markedly suppresses compound eye degeneration and inclusion body formation in a Drosophila model of SCA. 17-AAG also dramatically rescued the lethality of the SCA model (74.1% rescue) and suppressed neurodegeneration in a Huntington disease model (46.3% rescue), indicating that 17-AAG is widely effective against various polyQ diseases. 17-AAG induced Hsp70, Hsp40, and Hsp90 expression in a dose-dependent manner, and the expression levels correlated with its therapeutic effects. Furthermore, knockdown of HSF1 abolished the induction of molecular chaperones and the therapeutic effect of 17-AAG, indicating that its therapeutic effects depend on HSF1 activation. Our study indicates that induction of multiple molecular chaperones by 17-AAG treatment is a promising therapeutic approach for a wide range of polyQ diseases and possibly other neurodegenerative diseases.

VPS35 dysfunction impairs lysosomal degradation of α-synuclein and exacerbates neurotoxicity in a Drosophila model of Parkinson's disease

Mutations in vacuolar protein sorting 35 (VPS35) have been linked to familial Parkinsons disease (PD). VPS35, a component of the retromer, mediates the retrograde transport of cargo from the endosome to the trans-Golgi network. Here we showed that retromer depletion increases the lysosomal turnover of the mannose 6-phosphate receptor, thereby affecting the trafficking of cathepsin D (CTSD), a lysosome protease involved in α-synuclein (αSYN) degradation. VPS35 knockdown perturbed the maturation step of CTSD in parallel with the accumulation of αSYN in the lysosomes. Furthermore, we found that the knockdown of Drosophila VPS35 not only induced the accumulation of the detergent-insoluble αSYN species in the brain but also exacerbated both locomotor impairments and mild compound eye disorganization and interommatidial bristle loss in flies expressing human αSYN. These findings indicate that the retromer may play a crucial role in αSYN degradation by modulating the maturation of CTSD and might thereby contribute to the pathogenesis of the disease.

Detection of Polyglutamine Protein Oligomers in Cells by Fluorescence Correlation Spectroscopy

Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble β-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.

Induction of Molecular Chaperones as a Therapeutic Strategy for the Polyglutamine Diseases

Protein misfolding and aggregation in the brain have been implicated as a common molecular pathogenesis of various neurodegenerative diseases including Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and the polyglutamine (polyQ) diseases. The polyQ diseases are a group of nine hereditary neurodegenerative diseases, including Huntingtons disease (HD) and various types of spinocerebellar ataxia (SCA), which are caused by abnormal expansions of the polyQ stretch (> 35-40 repeats) in unrelated disease-causative proteins. The expanded polyQ stretch is thought to trigger misfolding of these proteins, leading to their aggregation and accumulation as inclusion bodies in affected neurons, eventually resulting in neurodegeneration. Misfolding and aggregation of the polyQ protein are the most ideal therapeutic targets since they are the most upstream events in the pathogenic cascade, and therefore, therapeutic approaches using molecular chaperones, which prevent protein misfolding and assist the refolding of misfolded proteins, are being extensively investigated. Indeed, a variety of molecular chaperones such as Hsp70 and Hsp40 have been demonstrated to exert therapeutic effects against various experimental models of the polyQ diseases. Furthermore, toward developing pharmacological therapies, small chemical activators of heat shock transcription factor 1 (HSF1) such as geldanamycin and its derivative 17-AAG, which induce multiple endogenous molecular chaperones, have been proven to be effective not only in polyQ disease models, but also in other neurodegenerative disease models. We hope that brain-permeable molecular chaperone inducers will be developed as drugs against a wide range of neurodegenerative diseases in the near future.

Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy

Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level

Significance The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is known to be activated in a cell type-specific manner. Despite such imbalanced HSR upon stress, it is unclear as to how organismal protein homeostasis (proteostasis) is maintained. Here, we show that elevated expression of molecular chaperones in cells non-cell autonomously improves proteostasis in other cells. We further show that exosome-mediated secretion and intercellular transmission of chaperones are responsible for this non–cell-autonomous improvement of proteostasis. Our study reveals a molecular mechanism of non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced HSR among different cells, and also provides a novel physiological function of exosomes that contributes to maintenance of proteostasis. The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non–cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis.

Alternative splicing regulates the transcriptional activity of Drosophila heat shock transcription factor in response to heat/cold stress.

Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock proteins in response to various types of stress. Although HSF1 activity is regulated by its post‐translational modifications, alterations in mRNA expression have also been suggested. We here identified three new alternatively spliced isoforms of Drosophila HSF (dHSF) mRNA, named dHSFb, dHSFc, and dHSFd. We found that the ratio of dHSFb increases upon heat exposure, while that of dHSFd increases upon cold exposure. The dHSFc and dHSFd isoforms showed greater transcriptional activity than the other isoforms. Our findings suggest that alternative splicing regulates the transcriptional activity of dHSF.

Knockdown of the Drosophila fused in sarcoma (FUS) homologue causes deficient locomotive behavior and shortening of motoneuron terminal branches.

Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. Here, we generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. Our results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model we presented recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS.

Delivery of the aggregate inhibitor peptide QBP1 into the mouse brain using PTDs and its therapeutic effect on polyglutamine disease mice.

The polyglutamine (polyQ) diseases are neurodegenerative diseases caused by proteins with an abnormally expanded polyQ stretch, which triggers abnormal aggregation of these proteins in the brain. We previously showed that the polyQ-binding peptide QBP1 inhibits polyQ aggregation, and further that administration of QBP1 fused with a protein transduction domain (PTD) suppresses polyQ-induced neurodegeneration in Drosophila. As the next step towards developing a therapy using QBP1, we investigated the delivery of PTD-QBP1 to the mouse brain upon its administration. Here we successfully detected delivery of PTD-QBP1 into mouse brain cells upon its single intracerebroventricular injection. In addition, long-term administration of PTD-QBP1 to polyQ disease mice improved their weight loss phenotype, suggesting a possible therapeutic effect. Our study indicates the potential of PTD-mediated delivery of QBP1 as a therapeutic strategy for the currently untreatable polyQ diseases.