Search

Abstract 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.

Abstract Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5′ cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.

Aggregation of α-synuclein (αSyn) and its accumulation as Lewy bodies play a central role in the pathogenesis of Parkinson’s disease (PD). However, the mechanism by which αSyn aggregates in the brain remains unclear. Biochemical studies have demonstrated that αSyn interacts with lipids, and these interactions affect the aggregation process of αSyn. Furthermore, genetic studies have identified mutations in lipid metabolism-associated genes such as glucocerebrosidase 1 (GBA1) and synaptojanin 1 (SYNJ1) in sporadic and familial forms of PD, respectively. In this review, we focus on the role of lipids in triggering αSyn aggregation in the pathogenesis of PD and propose the possibility of modulating the interaction of lipids with αSyn as a potential therapy for PD.

Abstract Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) has recently been attributed to biallelic repeat expansions in RFC1. More recently, the disease entity has expanded to atypical phenotypes, including chronic neuropathy without cerebellar ataxia or vestibular areflexia. Very recently, RFC1 expansions were found in patients with Sjögren syndrome who had neuropathy that did not respond to immunotherapy. In this study RFC1 was examined in 240 patients with acute or chronic neuropathies, including 105 with Guillain-Barré syndrome or Miller Fisher syndrome, 76 with chronic inflammatory demyelinating polyneuropathy, and 59 with other types of chronic neuropathy. Biallelic RFC1 mutations were found in three patients with immune-mediated neuropathies, including Guillain-Barré syndrome, idiopathic sensory ataxic neuropathy, or anti-myelin-associated glycoprotein (MAG) neuropathy, who responded to immunotherapies. In addition, a patient with chronic sensory autonomic neuropathy had biallelic mutations, and subclinical changes in Schwann cells on nerve biopsy. In summary, we found CANVAS-related RFC1 mutations in patients with treatable immune-mediated neuropathy or demyelinating neuropathy.

Abstract Background Huntington’s disease is an autosomal dominant inherited disorder characterized by personality changes (such as irritability and restlessness) and psychotic symptoms (such as hallucinations and delusions). When the personality changes become noticeable, involuntary movements (chorea) also develop. The disease is caused by the CAG repeat expansion in the coding region of the HTT gene, and the diagnosis is based on the presence of this expansion. However, there is currently no effective treatment for the progression of Huntington’s disease and its involuntary motor symptoms. Herein, we present a case in which memantine was effective in treating the chorea movements of Huntington’s disease. Case presentation A 75-year-old Japanese woman presented to the hospital with involuntary movements of Huntington’s disease that began when she was 73 years old. In a cerebral blood flow test (N-isopropyl-p-iodoamphetamine–single-photon emission computed tomography), decreased blood flow was observed in the precuneus (anterior wedge) and posterior cingulate gyrus. Usually, such areas of decreased blood flow are observed in patients with Alzheimer’s-type dementia. So, we administered memantine for Alzheimer’s-type dementia, and this treatment suppressed the involuntary movements of Huntington’s disease, and the symptoms progressed slowly for 7 years after the onset of senility. In contrast, her brother died of complications of pneumonia during the course of Huntington’s disease. Conclusions We recorded changes in parameters such as the results of the N-isopropyl-p-iodoamphetamine–single-photon emission computed tomography and gait videos over 7 years. Treatment with memantine prevented the chorea movement and the progression of Huntington’s disease. We believe this record will provide clinicians with valuable information in diagnosing and treating Huntington’s disease.

Dysphagia, a potentially fatal symptom of Parkinson's disease, is characterized by frequent silent aspiration, a risk factor for aspiration pneumonia. The transdermal dopamine agonist rotigotine alleviates dysphagia in patients with Parkinson's disease and is more effective than oral levodopa, suggesting the importance of continuous dopaminergic stimulation during swallowing. Rasagiline is a monoamine oxidase B (MAOB) inhibitor that facilitates continuous dopaminergic stimulation. We hypothesized that MAOB inhibition by rasagiline would be effective in improving swallowing function in patients with early- and mid-to late-stage Parkinson's disease. To this end, we performed an analytical observational study to determine the effects of rasagiline (1 mg/day) on swallowing function using videofluoroscopic swallowing study. This open-label, evaluator-blinded study enrolled 32 patients with Parkinson's disease, among whom 19 were drug-naïve and 13 were receiving add-on therapy. Our results showed that rasagiline significantly improved all swallowing measures during the oral and pharyngeal phases, including oral transit time and pharyngeal transit time, in all enrolled patients. Similar results were found in drug-naïve and mid-to late-stage patients, with no intergroup differences. In conclusion, drugs capable of continuous dopaminergic stimulation may effectively improve swallowing function in patients with Parkinson's disease, with similar effects in early- and mid-to late-stage Parkinson's disease. This study has been the first to show that rasagiline significantly improves swallowing function in mid-to late-stage patients receiving add-on therapy.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although how TDP-43 forms cytoplasmic aggregates and causes neurodegeneration in patients with ALS/FTD remains unclear, reducing cellular TDP-43 levels is likely to prevent aggregation and to rescue neurons from TDP-43 toxicity. To address this issue, here we developed gapmer-type antisense oligonucleotides (ASOs) against human TDP-43 using 2′-O,4′-C-ethylene nucleic acids (ENAs), which are modified nucleic acids with high stability, and tested the therapeutic potential of lowering TDP-43 levels using ENA-modified ASOs. We demonstrated that intracerebroventricular administration of ENA-modified ASOs into a mouse model of ALS/FTD expressing human TDP-43 results in the efficient reduction of TDP-43 levels in the brain and spinal cord. Surprisingly, a single injection of ENA-modified ASOs into TDP-43 mice led to long-lasting improvement of behavioral abnormalities and the suppression of cytoplasmic TDP-43 aggregation, even after TDP-43 levels had returned to the initial levels. Our results demonstrate that transient reduction of TDP-43 using ENA-modified ASOs leads to sustained therapeutic benefits in vivo, indicating the possibility of a disease-modifying therapy by lowering TDP-43 levels for the treatment of the TDP-43 proteinopathies, including ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Abstract Parkinson’s disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson’s disease.

Dysphagia is a potentially fatal symptom of Parkinson's disease (PD) and is characterized by frequent silent aspiration, a risk factor for aspiration pneumonia. The transdermal dopamine agonist rotigotine alleviates dysphagia in patients with PD and is more effective than oral levodopa, suggesting the importance of continuous dopaminergic stimulation (CDS) in swallowing. Safinamide is a monoamine oxidase B (MAOB) inhibitor that facilitates CDS. In this retrospective open-label evaluator-blinded research, swallowing functions in nine patients with PD were examined using a video fluoroscopic swallowing study (VFSS) before and after treatment with 50 mg of oral safinamide. The VFSS results showed that safinamide significantly improved some swallowing measures during oral and pharyngeal phases, including oral transit time and pharyngeal transit time, without worsening of any measures. Notably, improvements in lip closure, an oral phase component, seemed to be most attributable to improvements in oral phase scores. In conclusion, a medicine for CDS may effectively improve swallowing functions in patients with PD. This is the first study to show that the MAOB inhibitor safinamide partly but significantly improves swallowing function in patients with PD.

Seiya Oura and Taichi Noda et al. overcome the challenge of gene editing in CAG repeats, such as those causing Huntington’s Disease, using their recently developed SpCas9-NG variant. They demonstrate that SpCas9-NG can precisely edit and contract the CAG repeat tracks in a Huntington’s Disease mouse model, opening new avenues for research in this disease.

Abstract Background Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here the authors show the mechanism by which a small molecule targets the disease-causing r(UGGAA) n repeat RNAs in spinocerebellar ataxia type 31.

Extracellular vesicles (EVs) have attracted much attention as potential diagnostic biomarkers for human diseases. Although both plasma and serum are utilized as a source of blood EVs, it remains unclear whether, how and to what extent the choice of plasma and serum affects the experimental results. To address this issue, in this study, we performed comprehensive characterization of EV fractions derived from plasma and serum, and investigated the differences between these blood EVs. We demonstrated by nanoparticle tracking analysis that EV fractions derived from serum contain more particles than those from plasma of mice. Proteomic analysis demonstrated that platelet-associated proteins are selectively enriched in serum EV fractions from both mice and humans. A literature review of proteomic data of human blood EVs reported by other groups further confirmed that selective enrichment of platelet-associated proteins is commonly observed in serum EVs, and confers different proteome profiles to plasma EVs. Our data provide experimental evidence that EV fractions derived from serum generally contain additional EVs that are released from platelets, which may qualitatively and quantitatively alter EV profiles when using serum as a source of blood EVs.

Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 (BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease–linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.

The non-coding GGGGCC hexanucleotide repeat expansion (HRE) in C9orf72 gene is a dominant cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This intronic mutation elicits the formation of nuclear and cytoplasmic inclusions containing RNA, RNA-binding proteins, and HRE-derived dipeptide repeat proteins (DPRs), leading to neurodegeneration via the gain-of-toxic function or loss-of-function of relevant proteins. Using C9-500 mice harboring ~500 repeats of the GGGGCC sequence in human C9orf72 gene, we investigated the effects of rifampicin against HRE-related pathological phenotypes. Rifampicin was administered intranasally to 4.5- to 5-month-old mice for 1 month, and their cognitive function and neuropathology were assessed by the Morris water maze test and immunohistochemical staining. Rifampicin treatment reduced the formation of RNA foci and cytoplasmic inclusions containing DPRs or phosphorylated TDP-43, and furthermore, the levels of phosphorylated double-strand RNA-dependent protein kinase (PKR) that regulates repeat-associated non-ATG (RAN) translation. Synapse loss in the hippocampus and neuronal loss and microglial activation in the prefrontal and motor cortices were also attenuated, and mouse memory was significantly improved. Our findings suggest a therapeutic potential of nasal rifampicin in the prevention of C9orf72-linked neurodegenerative disorders.

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases caused by the abnormal expansion of a CAG trinucleotide repeat that are translated into an expanded polyQ stretch in the disease-causative proteins. The expanded polyQ stretch itself plays a critical disease-causative role in the pathomechanisms underlying polyQ diseases. Notably, the expanded polyQ stretch undergoes a conformational transition from the native monomer into the β-sheet-rich monomer, followed by the formation of soluble oligomers and then insoluble aggregates with amyloid fibrillar structures. The intermediate soluble species including the β-sheet-rich monomer and oligomers exhibit substantial neurotoxicity. Therefore, protein conformation stabilization and aggregation inhibition that target the upstream of the insoluble aggregate formation would be a promising approach toward the development of disease-modifying therapies for polyQ diseases. PolyQ aggregation inhibitors of different chemical categories, such as intrabodies, peptides, and small chemical compounds, have been identified through intensive screening methods. Among them, recent advances in the brain delivery methods of several peptides and the screening of small chemical compounds have brought them closer to clinical utility. Notably, the recent discovery of arginine as a potent conformation stabilizer and aggregation inhibitor of polyQ proteins both in vitro and in vivo have paved way to the clinical trial for the patients with polyQ diseases. Meanwhile, expression reduction of expanded polyQ proteins per se would be another promising approach toward disease modification of polyQ diseases. Gene silencing, especially by antisense oligonucleotides (ASOs), have succeeded in reducing the expression of polyQ proteins in the animal models of various polyQ diseases by targeting the aberrant mRNA with expanded CAG repeats. Of note, some of these ASOs have recently been translated into clinical trials. Here we overview and discuss these recent advances toward the development of disease modifying therapies for polyQ diseases. We envision that combination therapies using aggregation inhibitors and gene silencing would meet the needs of the patients with polyQ diseases and their caregivers in the near future to delay or prevent the onset and progression of these currently intractable diseases.

Animal models are indispensable tools in the development of innovative treatments for rare and incurable diseases. To date, there is almost no effective treatment for neurodegenerative diseases, and animal models that properly simulate human disease pathologies are eagerly anticipated to identify disease biomarkers and develop therapeutic methods and agents. Among experimental animals, non-human primates are the most suitable animal models for the study of neurodegenerative diseases with human-specific higher brain dysfunction and late-onset and slowly progressing symptoms. With the rapid development of novel therapies such as oligonucleotide therapeutics and genome editing technologies, non-human primate models for neurodegenerative diseases will be essential for preclinical studies and active interventional trials. In a previous publication, we reported the generation of the first transgenic marmoset model of spinocerebellar ataxia type 3 and successful obtainment of subsequent generations with stable disease onset. Moreover, we generated transgenic marmosets in which the transgene was controlled by the tetracycline-inducible gene expression system. In this mini-review, we summarize the research on our marmoset model of spinocerebellar ataxia type 3.

α-Synuclein (αSyn) plays a major role in the pathogenesis of Parkinson’s disease (PD), which is the second most common neurodegenerative disease after Alzheimer’s disease. The accumulation of αSyn is a pathological hallmark of PD, and mutations in the SNCA gene encoding αSyn cause familial forms of PD. Moreover, the ectopic expression of αSyn has been demonstrated to mimic several key aspects of PD in experimental model systems. Among the various model systems, Drosophila melanogaster has several advantages for modeling human neurodegenerative diseases. Drosophila has a well-defined nervous system, and numerous tools have been established for its genetic analyses. The rapid generation cycle and short lifespan of Drosophila renders them suitable for high-throughput analyses. PD model flies expressing αSyn have contributed to our understanding of the roles of various disease-associated factors, including genetic and nongenetic factors, in the pathogenesis of PD. In this review, we summarize the molecular pathomechanisms revealed to date using αSyn-expressing Drosophila models of PD, and discuss the possibilities of using these models to demonstrate the biological significance of disease-associated factors.

Huntington’s disease (HD) is an intractable neurodegenerative disorder caused by mutant Huntingtin (HTT) proteins that adversely affect various biomolecules and genes. MicroRNAs (miRNAs), which are functional small non-coding RNAs, are also affected by mutant HTT proteins. Here, we show amelioration in motor function and lifespan of HD-model mice, R6/2 mice, by supplying miR-132 to HD brains using a recombinant adeno-associated virus (rAAV) miRNA expression system. miR-132 is an miRNA related to neuronal maturation and function, but the level of miR-132 in the brain of R6/2 mice was significantly lower than that of wild-type mice. Our miR-132 supplemental treatment, i.e., supplying miR-132 to the brain, produced symptomatic improvement or retarded disease progression in R6/2 mice; interestingly, it had little effect on disease-causing mutant HTT mRNA expression and its products. Therefore, the findings suggest that there may be a therapeutic way to treat HD without inhibiting and/or repairing disease-causing HTT genes and gene products. Although miR-132 supplement may not be a definitive treatment for HD, it may become a therapeutic method for relieving HD symptoms and delaying HD progression.

Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Lewy bodies (LBs) and Lewy neurites, which consist mainly of aggregated αSyn, are widely observed in the affected regions of patient brains. Except for some familial forms of PD/DLB, most sporadic PD/DLB patients express the wild-type (WT) αSyn protein without any mutations, and the mechanisms as to how WT αSyn gains the propensity to pathologically aggregate still remains unclear. Furthermore, the mechanisms by which the same αSyn protein can cause different synucleinopathies with distinct phenotypes and pathologies, such as PD, DLB, and multiple system atrophy (MSA), still remain largely unknown. Recently, mutations in the GBA1 gene (encoding glucocerebrosidase), which are responsible for the lysosomal storage disorder Gaucher disease (GD), have been reported to be the strongest risk factor for developing sporadic PD/DLB. We previously demonstrated that glucosylceramide accumulated by GBA1 deficiency promotes the conversion of αSyn into a proteinase K-resistant conformation. Furthermore, decreased glucocerebrosidase activity has also been reported in the brains of patients with sporadic PD/DLB. Moreover, αSyn pathology has also been shown in the brains of lysosomal storage disorder patients, which show glycosphingolipid accumulation. These observations suggest the possibility that altered lipid metabolism and lipid accumulation play roles in αSyn aggregation and PD/DLB pathogenesis. Indeed, several previous studies have demonstrated that lipid interactions affect the conformation of αSyn and induces its oligomerization and aggregation. In this review, we will give an overview of the association between αSyn aggregation and lipid interactions from the viewpoints of the etiology, pathology, and genetics of PD/DLB. We also discuss the distinct species of αSyn aggregates and their association with specific types of synucleinopathies, and introduce our hypothesis that lipid interactions play a role as trans-acting effectors in producing distinct strains of αSyn fibrils.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by progressive motor dysfunction as well as non-motor symptoms. Pathological and genetic studies have demonstrated that α-synuclein (αSyn) plays key roles in the pathogenesis of PD. Although several missense mutations in the αSyn gene have been identified as causes of familial PD, the mechanisms underlying the variance in the clinical phenotypes of familial PD caused by different mutations remain elusive. Here, we established novel Drosophila models expressing either wild-type (WT) αSyn or one of five αSyn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant αSyn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these αSyn-expressing flies showed a progressive decline in locomotor function. Notably, we found that E46K, H50Q, G51D, and A53T αSyn-expressing flies showed earlier onset of locomotor dysfunction than WT αSyn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant αSyn were almost equivalent, we found that protein expression levels of E46K αSyn were higher than those of WT αSyn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K αSyn protein was significantly slower than WT αSyn protein, indicating that the E46K αSyn mutant gains resistance to degradation in vivo. We therefore conclude that our novel site-specific transgenic fly models expressing either WT or mutant αSyn are useful to explore the mechanisms by which different αSyn mutants gain toxic functions in vivo.

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.

Sleep disturbance is a common symptom in patients with various neurodegenerative diseases, including Alzheimer’s disease (AD), and it can manifest in the early stages of the disease. Impaired sleep in patients with AD has been attributed to AD pathology that affects brain regions regulating the sleep⁻wake or circadian rhythm. However, recent epidemiological and experimental studies have demonstrated an association between impaired sleep and an increased risk of AD. These studies have led to the idea of a bidirectional relationship between AD and impaired sleep; in addition to the conventional concept that impaired sleep is a consequence of AD pathology, various evidence strongly suggests that impaired sleep is a risk factor for the initiation and progression of AD. Despite this recent progress, much remains to be elucidated in order to establish the benefit of therapeutic interventions against impaired sleep to prevent or alleviate the disease course of AD. In this review, we provide an overview of previous studies that have linked AD and sleep. We then highlight the studies that have tested the causal relationship between impaired sleep and AD and will discuss the molecular and cellular mechanisms underlying this link. We also propose future works that will aid the development of a novel disease-modifying therapy and prevention of AD via targeting impaired sleep through non-pharmacological and pharmacological interventions.

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.

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.

The polyglutamine (polyQ) diseases, such as Huntington’s disease and several types of spinocerebellar ataxias, are a group of inherited neurodegenerative diseases that are caused by an abnormal expansion of the polyQ tract in disease-causative proteins. Proteins with an abnormally expanded polyQ stretch undergo a conformational transition to β-sheet rich structure, which assemble into insoluble aggregates with β-sheet rich amyloid fibrillar structures and accumulate as inclusion bodies in neurons, eventually leading to neurodegeneration. Since misfolding and aggregation of the expanded polyQ proteins are the most upstream event in the most common pathogenic cascade of the polyQ diseases, they are proposed to be one of the most ideal targets for development of disease-modifying therapies for polyQ diseases. In this review, we summarize the current understanding of the molecular pathogenic mechanisms of the polyQ diseases, and introduce therapeutic approaches targeting misfolding and aggregation of the expanded polyQ proteins, which are not only effective on a wide spectrum of polyQ diseases, but also broadly correct the functional abnormalities of multiple downstream cellular processes affected in the aggregation process of polyQ proteins. We hope that in the near future, effective therapies are developed, to bring hope to many patients suffering from currently intractable polyQ diseases.

A new methodology has been developed to determine sphingolipid structures by positive-ion fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The method was verified by application to a structurally known glycosphingolipid, and then used in the structural study of an unusual sphingomyelin isolated from squid (Loligo pealei) nerve. Our previous study of this squid sphingomyelin indicated that the major base had a branched C19 alkyl chain with three double bonds, two of which were conjugated. The positions of the branching as well as the double bonds of this base were unambiguously determined by directly comparing the product ion spectra of the long-chain base ion (LCB+) of two ceramides, one derived from squid nerve sphingomyelin and another, glucosylceramide, obtained from starfish spermatozoa. The latter served as the standard because the structure had already been determined by nuclear magnetic resonance (NMR). The precursor ion here was LCB+, that is, [CH2 − C(NH2) = CHR]+, rather than [M + H]+, where R represents the backbone hydrocarbon chain counting from C-4. The results clearly showed that the squid nerve base is identical to the base derived from starfish (Asterias amurensis), that is, 2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol.This is the first report in which the detailed structure of a branched polyunsaturated sphingoid base was studied by tandem mass spectrometry without derivatization or the aid of NMR. The occurrence of such an unusual sphingoid base in various phyla and tissues suggests the conjugated polyunsaturated branched sphingoid base plays a significant role in animals.

In polyglutamine diseases, an abnormally elongated polyglutamine results in protein misfolding and accumulation of intracellular aggregates. Autophagy is a major cellular degradative pathway responsible for eliminating unnecessary proteins, including polyglutamine aggregates. Basal autophagy constitutively occurs at low levels in cells for the performance of homeostatic function, but the regulatory mechanism for basal autophagy remains elusive. Here we show that the Na(+)/H(+) exchanger (NHE) family of ion transporters affect autophagy in a neuron-like cell line (Neuro-2a cells). We showed that expression of NHE1 and NHE5 is correlated to polyglutamine accumulation levels in a cellular model of Huntington's disease, a fatal neurodegenerative disorder characterized by accumulation of polyglutamine-containing aggregate formation in the brain. Furthermore, we showed that loss of NHE5 results in increased polyglutamine accumulation in an animal model of Huntington's disease. Our data suggest that cellular pH regulation by NHE1 and NHE5 plays a role in regulating basal autophagy and thereby promotes autophagy-mediated degradation of proteins including polyglutamine aggregates.

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.

The polyglutamine (polyQ) diseases such as Huntington's disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.

α-Synuclein (α-Syn) is a major component of protein inclusions known as Lewy bodies, which are hallmarks of synucleinopathies such as Parkinson's disease (PD). The α-Syn gene is one of the familial PD-causing genes and is also associated with an increased risk of sporadic PD. Numerous studies using α-Syn expressing transgenic animals have indicated that α-Syn plays a critical role in the common pathogenesis of synucleinopathies. Drosophila melanogaster has several advantages for modeling human neurodegenerative diseases and is widely used for studying their pathomechanisms and therapies. In fact, Drosophila models expressing α-Syn have already been established and proven to replicate several features of human PD. In this paper, we review the current research on synucleinopathies using α-Syn Drosophila models and, moreover, explore the possibilities of these models for comprehensive genetic analyses and large-scale drug screening towards elucidating the molecular pathogenesis and developing therapies for synucleinopathies.

A technique was developed to isolate sufficient material for compositional analysis of cerebroside from pooled human cerebrospinal fluid. The carbohydrate moiety was principally galactose. The sphingosine base and fatty acid compositions were found to be similar to that of brain cerebroside. The presence of a contaminant in commercial silica gel which chromatographed like the trimethylsilyl derivative of glucose is described.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy. [ABSTRACT FROM AUTHOR]