Mari Suzuki

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.

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.

Aversive taste stimuli facilitate extracellular acetylcholine release in the insular gustatory cortex of the rat : a microdialysis study

The release of extracellular acetylcholine (ACh) in the insular gustatory cortex of conscious rats during taste stimulation was measured using the microdialysis technique. The mean basal release of ACh before stimulation was 273 +/- 21 fmol/10 microliters (mean +/- S.E.M., n = 25). Intraorally applied taste stimuli or distilled water significantly increased the release of ACh. Among them, infusion of 0.001 M quinine HCl produced a marked increase in the release of ACh up to 355% of baseline levels. Infusion of 0.01 M saccharin to the subjects that had acquired an aversion to this taste also caused a prominent increase in ACh up to 343% of basal levels. In contrast, saccharin infusion to the naive subjects moderately increased ACh up to 243% of baseline. Water infusion resulted in the smallest increase in ACh up to 175% of baseline. Although intraoral infusions of quinine or distilled water caused a significant increase in ACh in the parietal cortex, the magnitude of increased ACh was smaller than that in the gustatory cortex. These results suggest that ACh release in the insular gustatory cortex is related to behavioral expression to aversive taste stimuli.

Molecular Basis of Orb2 Amyloidogenesis and Blockade of Memory Consolidation

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant α-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease

Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gauchers disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. In this study, we employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses showed that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation was associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments revealed that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of β-galactosidase (β-Gal) also aggravated locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerated the formation of PK-resistant αSyn. Our findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

Calcium leak through ryanodine receptor is involved in neuronal death induced by mutant huntingtin

Huntingtons disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine (polyQ) tract in huntingtin (htt) protein. Although altered calcium (Ca(2+)) homeostasis is suggested in HD, its molecular mechanisms have remained poorly understood despite their important role in the pathogenesis. In this study, we examined involvement of ryanodine receptor (RyR), an endoplasmic reticulum-resident Ca(2+) channel, in mutant htt-induced neuronal death. Inhibitors of RyR attenuated cell death induced by mutant htt, while co-expression of RyR enhanced htt toxicity. Intracellular Ca(2+) imaging revealed that mutant htt caused excessive basal Ca(2+) release (Ca(2+) leak) through RyR leading to depletion of internal Ca(2+) store. Ca(2+) leak was also observed in striatal and cortical neurons from R6/2 HD model mice. Moreover, expression of FK506-binding protein 12 (FKBP12), a RyR stabilizer, suppressed both Ca(2+) leak and cell death. These results provide novel evidence suggesting altered RyR function is involved in neuronal cell death, and its stabilization might be beneficial for treatment of HD.

P62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies

Background: Oligomers of pathogenic proteins are implicated in the pathomechanisms of neurodegenerative diseases. Results: Depletion of p62 delays the degradation of polyglutamine protein oligomers via autophagy and exacerbates neurodegeneration in polyglutamine disease model flies. Conclusion: p62 plays a protective role via autophagic degradation of polyglutamine protein oligomers. Significance: p62 should be a therapeutic target for the polyglutamine diseases. Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. In this study, to elucidate the role of p62 in such pathogenic conditions in vivo, we used Drosophila models of neurodegenerative diseases. We found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. We also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. We therefore conclude that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases.

Ubiquitin Carboxyl-Terminal Hydrolase L3 Promotes Insulin Signaling and Adipogenesis

Insulin is a potent adipogenic hormone that triggers the induction of a series of transcription factors and specific proteins governing the differentiation of preadipocytes into mature adipocytes. Here we report that ubiquitin carboxyl-terminal hydrolase (UCH)-L3, a deubiquitinating enzyme, promotes insulin signaling and adipogenesis. Uchl3(-/-) mice had less visceral white adipose tissue compared with wild-type mice. In vitro adipogenesis experiments revealed that mouse embryonic fibroblasts (MEFs) and preadipocytes from Uchl3(-/-) mice had impaired ability to differentiate into mature adipocytes than those from wild-type mice. This difference was diminished by removing insulin from the medium. RT-PCR analysis showed that insulin-regulated expression of srebp1c, fas, glut4, and adiponectin is impaired in Uchl3(-/-) cells. The phosphorylation of insulin/IGF-I receptor, Akt, glycogen synthase kinase-3beta, and FoxO1 was decreased in Uchl3(-/-) MEFs treated with insulin. Moreover, ectopic expression of wild-type UCH-L3 restored the phosphorylation of insulin/IGF-I receptor and adipocyte differentiation in Uchl3(-/-) MEFs. In contrast, hydrolase activity-deficient UCH-L3 did not enhance insulin signaling and the expression of glut4, fabp4, and adiponectin, resulting in impaired formation of large lipid droplets. These results suggest that UCH-L3 promotes adipogenesis by enhancing insulin signaling in a hydrolase activity-dependent manner.

Ubiquitin C-terminal hydrolase-L3-knockout mice are resistant to diet-induced obesity and show increased activation of AMP-activated protein kinase in skeletal muscle

Obesity results from the dysregulation of energy balance throughout the entire body. Although the ubiquitin system participates in many cellular pro‐ cesses, its contribution to the balance of energy in the body remains poorly understood. Here, we show that ubiquitin C‐terminal hydrolase (UCH)‐L3, one of the deubiquitinating enzymes, contributes to the regulation of metabolism. Uchl3−/− mice displayed a reduction of adipose tissue mass and were protected against high‐fat diet (HFD)‐induced obesity and insulin resistance. Uchl3−/− mice given both a normal chow and an HFD had an increased whole‐body energy expenditure ac‐ counting for the reduction of adipose tissue mass. Activation of AMP‐activated protein kinase (AMPK) in skeletal muscle has been reported to increase fatty acid β‐oxidation, leading to the elevation of the whole‐body energy expenditure. Consistently, increased activation of AMPK and fatty acid β‐oxidation was observed in skeletal muscle of Uchl3−/− mice. Mouse embryonic fibroblasts derived from Uchl3−/− mice also showed increased activation of AMPK, indicating that UCH‐L3 is involved in a cell‐autonomous down‐regulation of AMPK. These results suggest a role for UCH‐L3 in the regulation of AMPK activity and whole‐body energy metabolism.—Setsuie, R., Suzuki, M., Kabuta, T., Fu‐ jita, H., Miura, S., Ichihara, N., Yamada, D., Wang, Y.‐L., Ezaki, O., Suzuki, Y., Wada, K. Ubiquitin C‐ terminal hydrolase‐L3‐knockout mice are resistant to diet‐induced obesity and show increased activation of AMP‐activated protein kinase in skeletal muscle. FASEB J. 23, 4148‐4157 (2009). www.fasebj.org

Skeletal muscles of Uchl3 knockout mice show polyubiquitinated protein accumulation and stress responses

Ubiquitin C-terminal hydrolase (UCH)-L3 is an enzyme with a strongly suggested de-ubiquitinating function by in vitro studies, but has poorly been investigated in vivo. In this study, we show that skeletal muscles of Uchl3(-/-) mice exhibit the up-regulation of cleaved ATF6, Grp78, and PDI as well as HSP27, HSP70, HSP90 and HSP110, which indicate the induction of stress responses. The prominent accumulation of polyubiquitinated proteins, one of the factors reported to induce stress responses, was observed in the skeletal muscle of Uchl3(-/-) mice. Mouse embryonic fibroblasts (MEFs) from Uchl3(-/-) mice also showed an accumulation of polyubiquitinated proteins. Moreover, the polyubiquitinated protein accumulation in Uchl3(-/-) MEFs was attenuated by the exogenous expression of wild-type, but not hydrolase activity deficient, UCH-L3. In addition, wild-type, but not its hydrolase activity or ubiquitin binding activity deficient UCH-L3 showed the ability to cleave ubiquitin from polyubiquitinated lysozyme in vitro. These results suggest that UCH-L3 functions as a de-ubiquitinating enzyme in vivo where lack of its hydrolase activity may result in the prominent accumulation of ubiquitinated proteins and subsequent induction of stress responses in skeletal muscle.