Synthesis of Novel Polymers with Biodegradability by Main-Chain Editing of Chiral Polyketones.

Although the use of biodegradable plastics is suitable for unrecoverable, single-use plastic, their high production cost and much lower variety compared to commodity plastics limit their application. In this study, we developed a new polymer with potential biodegradability, poly(ketone/ester), synthesized from propylene and carbon monoxide. Propylene and carbon monoxide are easily available at low costs from fossil resources, and they can also be derived from biomass. Using an atom insertion reaction to the main chain of the polymer, the main-chain editing of the polymer molecule proceeded with up to 89% selectivity for atom insertion over main-chain cleavage.

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

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.

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 Parkinson's 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 Gaucher's 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 ß-galactosidase (ß-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 ß-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

p62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies.

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.

Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS.

In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previously, we established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. Here, we aimed to explore molecules that affect these ALS-like phenotypes of our Drosophila models with eye-specific and neuron-specific Caz-knockdown. We examined several previously reported ALS-related genes and found genetic links between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. Our data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. Our results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS.

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 Parkinson's 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.

Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

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 beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-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.

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

Hypoglycosylation and reduced laminin-binding activity of alpha-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 alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-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 alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-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 alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

Surface plasmon resonance characterization of specific binding of polyglutamine aggregation inhibitors to the expanded polyglutamine stretch.

Proteins with an abnormally expanded polyglutamine (polyQ) stretch are prone to change their conformations, leading to their aggregation, and cause inherited neurodegenerative diseases called the polyQ diseases. Although screening for polyQ aggregation inhibitors has been extensively performed, many common false-positive hits have been identified so far. In this study, we employed surface plasmon resonance (SPR) to characterize the binding specificities and affinities of polyQ aggregation inhibitors to the expanded polyQ stretch. SPR successfully detected specific binding of polyQ binding peptide 1 (QBP1) to the expanded polyQ stretch (K(d)=5.7 microM), and non-specific binding of Congo red to polyQ proteins independent of their polyQ-length. Binding affinities of polyQ aggregation inhibitors to the expanded polyQ stretch were correlated with their inhibitory effects on polyQ aggregation. We therefore conclude that SPR is a useful technique for screening for specific polyQ aggregation inhibitors as promising therapeutic candidates for the currently untreatable polyQ diseases.

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.

ER stress is the initial response to polyglutamine toxicity in PC12 cells.

Persistent endoplasmic reticulum (ER) stress and impairment of the ubiquitin-proteasome system (UPS) cause neuronal cell death. However, the relationship between these two phenomena remains controversial. In our current study, we have utilized an expanded polyglutamine fusion protein (polyQ81) expression system in PC12 cells to further examine the involvement of ER stress and UPS impairment in cell death. The expression of polyQ81-induced ER stress and cell death. PolyQ81 also induced the activation of c-Jun N-terminal kinase (JNK) and caspase-3 and an increase in polyubiquitin immunoreactivity, suggesting UPS impairment. ER stress was induced prior to the accumulation of polyubiquitinated proteins. Low doses of lactacystin had almost similar effects on cell viability and on the activation of JNK and caspase-3 between normal cells and polyQ81-expressing cells. These results suggest that ER stress mediates polyglutamine toxicity prior to UPS impairment during the initial stages of these toxic effects.

Association Between Autophagy and Neurodegenerative Diseases.

Autophagy is a phylogenetically conserved mechanism that controls the degradation of subcellular constituents, including misfolded proteins, and damaged organelles. The progression of many neurodegenerative diseases is thought to be driven by the aggregation of misfolded proteins; therefore, autophagic activity is thought to affect disease severity to some extent. In some neurodegenerative diseases, the suppression of autophagic activity accelerates disease progression. Given that the induction of autophagy can potentially mitigate disease severity, various autophagy-inducing compounds have been developed and their efficacy has been evaluated in several rodent models of neurodegenerative diseases.

Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31.

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

Cytoprotective effect of novel histone deacetylase inhibitors against polyglutamine toxicity.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurological disorder caused by a CAG repeat expansion in the DRPLA gene encoding polyglutamine (polyQ). Although previous experimental studies have demonstrated that histone deacetylase (HDAC) inhibitors are therapeutically active, known HDAC inhibitors have considerable adverse effects clinically. To identify new HDAC inhibitors for the treatment of DRPLA, we evaluated a new series of HDAC inhibitors, N-hydroxycarboxamides, with our drug screening system, which uses neuronal PC12 cells stably transfected with a part of the DRPLA gene. We found that two of four N-hydroxycarboxamides significantly reduced polyQ-induced cell death. The essential structure of these compounds is a hydroxamic acid residue, which is shared with trichostatin A, a known HDAC inhibitor. Although our study showed mild neuroprotective effects, further structural modification of compounds that retain this residue may decrease cytotoxicity and increase protective activity against polyQ toxicity.

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.

Humanin attenuates apoptosis induced by DRPLA proteins with expanded polyglutamine stretches.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal-dominant neurodegenerative disorder caused by expansion of CAG repeats in the DRPLA gene, which codes for a polyglutamine (polyQ) stretch. The expanded polyQs are known to form intracellular aggregates and to confer neurotoxic activity. Recent studies have indicated that activation of apoptosis signal-regulating kinase 1 (ASK1) is involved in polyQ-induced apoptosis. Humanin (HN) is an endogenous peptide that inhibits neuronal cell death caused by mutant Alzheimer's disease genes, and this neuroprotective factor has recently been reported to suppress apoptosis by inhibiting activation of ASK1. To test the anti-ASK1 effect of HN on polyQ neurotoxicity, we constructed neuronal PC12 cells expressing expanded polyQs under the control of the Tet-Off system. Using this cell line, we showed that HN suppresses apoptotic cell death induced by expanded polyQs. However, the suppression was incomplete, suggesting that polyQs also stimulate other pathogenic cascades unrelated to ASK1. We further showed that HN suppresses polyQ aggregate formation. This result implied the possibility that aggregation is also related to the polyQ-mediated cascade involving ASK1 activation. Although the details remain uncertain, our results suggest that ASK1 is potentially involved in pathogenesis of DRPLA and that HN might contribute partially to the suppression of neurodegeneration in polyQ diseases.

Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference.

The α-synuclein (SNCA) gene is a responsible gene for Parkinson's disease (PD); and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi); however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs) that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named "expression-control RNAi" (ExCont-RNAi). ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.

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.