Arginine is a disease modifier for polyQ disease models that stabilizes polyQ protein conformation.

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal ß-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic ß-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.

Sleep Disturbance as a Potential Modifiable Risk Factor for Alzheimer's 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.

Differential recruitment of UBQLN2 to nuclear inclusions in the polyglutamine diseases HD and SCA3.

Accumulation of mutant polyglutamine proteins in intraneuronal inclusions is a hallmark of polyglutamine diseases. Impairment of protein clearance systems and sequestration of clearance-related proteins into inclusions occur in many protein folding diseases, including polyglutamine diseases. The ubiquitin-binding and proteasome adaptor protein UBQLN2 participates in protein homeostasis and localizes to inclusions in various neurodegenerative diseases. Employing mouse models and human brain tissue of Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3), we show that UBQLN2 is selectively recruited to inclusions in HD but not SCA3. Consistent with this result, in a cell-based system mutant HTT interacts with UBQLN2 through the UBA domain while the SCA3 disease protein ATXN3, a deubiquitinating enzyme, does not interact with UBQLN2. Differential recruitment of UBQLN2 to aggregates in HD and SCA3 underscores the heterogeneity of inclusions in polyglutamine diseases and suggests that components of neuronal protein quality control may be differentially perturbed in distinct polyQ diseases.

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.

Optimization of piggyBac transposon-mediated gene transfer method in common marmoset embryos.

Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.

Sustained therapeutic benefits by transient reduction of TDP-43 using ENA-modified antisense oligonucleotides in ALS/FTD mice.

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.

Bidirectional Relationship Between Sleep Disturbances and Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Both diseases share common clinical and pathological features: the gradual progression of neurological and psychiatric symptoms caused by neuronal dysfunction and neuronal cell death due to the accumulation of misfolded and neurotoxic proteins. Furthermore, both of them are multifactorial diseases in which both genetic and non-genetic factors contribute to the disease course. Non-genetic factors are of particular interest for the development of preventive and therapeutic approaches for these diseases because they are modifiable; of these, sleep is a particularly intriguing factor. Sleep disturbances are highly prevalent among both patients with AD and PD. To date, research has suggested that sleep disturbances are a consequence as well as a risk factor for the onset and progression of AD, which implies a bidirectional relationship between sleep and AD. Whether such a relationship exists in PD is less certain, albeit highly plausible given the shared pathomechanisms. This review examines the current evidence for the bidirectional relationship between sleep and PD. It includes research in both humans and animal models, followed by a discussion of the current understanding of the mechanisms underlying this relationship. Finally, potential avenues of research toward achieving disease modification to treat or prevent PD are proposed. Although further efforts are crucial for preventing the onset and slowing the progress of PD, it is evident that sleep is a valuable candidate target for future interventions to improve the outcomes of PD patients.

Protein Aggregation Inhibitors as Disease-Modifying Therapies for Polyglutamine Diseases.

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 ß-sheet-rich monomer, followed by the formation of soluble oligomers and then insoluble aggregates with amyloid fibrillar structures. The intermediate soluble species including the ß-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.

Human iPS cell-derived mural cells as an in vitro model of hereditary cerebral small vessel disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common forms of hereditary cerebral small vessel diseases and is caused by mutations in NOTCH3. Our group has previously reported incorporation of NOTCH3 extracellular domain (N3ECD) in the CADASIL-specific granular osmiophilic materials and increase of PDGFRß immunoreactivity in CADASIL postmortem brains. Here, we aimed to establish an in vitro model of CADASIL, which can recapitulate those CADASIL phenotypes, using induced pluripotent stem cells (iPSCs). We have refined a differentiation protocol of endothelial cells to obtain mature mural cells (MCs) with their characteristic properties. iPSCs from three CADASIL patients with p.Arg182Cys, p.Arg141Cys and p.Cys106Arg mutations were differentiated into MCs and their functional and molecular profiles were compared. The differentiated CADASIL MCs recapitulated pathogenic changes reported previously: increased PDGFRß and abnormal structure/distribution of filamentous actin network, as well as N3ECD/LTBP-1/HtrA1-immunopositive deposits. Migration rate of CADASIL MCs was enhanced but suppressed by knockdown of NOTCH3 or PDGFRB. CADASIL MCs showed altered reactivity to PDGF-BB. Patient-derived MCs can recapitulate CADASIL pathology and are therefore useful in understanding the pathogenesis and developing potential treatment strategies.

[Sleep abnormality as a potential target of disease-modifying therapy for neurodegenerative diseases].

Sleep abnormality such as frequent nocturnal arousal and decreased deep non-REM (rapid-eye-movement) sleep is a prevalent but under-recognized symptom that affects patients with various neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). In contrast to the conventional understanding that the sleep abnormality in these patients is caused by AD or PD pathology in the brain regions regulating sleep-wake or circadian rhythm, various epidemiological studies have demonstrated the association of sleep abnormality with an increased risk of these diseases. Through various recent studies using relevant animal models to test the causal relationship between sleep abnormality and neurodegenerative diseases, the recent concept of a bidirectional relationship between sleep abnormality and neurodegenerative diseases was established. However, whether therapeutic interventions against sleep abnormality would modify the disease course of neurodegenerative diseases remains unknown. In this review, we will first provide an overview of previous studies that link neurodegenerative diseases and sleep abnormality, mainly focusing on the sleep abnormality in patients with AD. We will then introduce the studies that examined the causal relationship between sleep abnormality and neurodegenerative diseases. Finally, we will discuss possible mechanisms underlying the bidirectional relationship between sleep abnormality and neurodegenerative diseases. A better understanding of these mechanisms would lead to the development of novel pharmacological and/or non-pharmacological treatments that would modify the disease course of neurodegenerative diseases through targeting the processes related to sleep abnormality in the patients of neurodegenerative diseases.

E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease.

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.

Chronic sleep fragmentation exacerbates amyloid ß deposition in Alzheimer's disease model mice.

Sleep fragmentation due to intermittent nocturnal arousal resulting in a reduction of total sleep time and sleep efficiency is a common symptom among people with Alzheimer's disease (AD) and elderly people with normal cognitive function. Although epidemiological studies have indicated an association between sleep fragmentation and elevated risk of AD, a relevant disease model to elucidate the underlying mechanisms was lacking owing to technical limitations. Here we successfully induced chronic sleep fragmentation in AD model mice using a recently developed running-wheel-based device and demonstrate that chronic sleep fragmentation increases amyloid ß deposition. Notably, the severity of amyloid ß deposition exhibited a significant positive correlation with the extent of sleep fragmentation. These findings provide a useful contribution to the development of novel treatments that decelerate the disease course of AD in the patients, or decrease the risk of developing AD in healthy elderly people through the improvement of sleep quality.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Chicken DT40 cell line lacking DJ-1, the gene responsible for familial Parkinson's disease, displays mitochondrial dysfunction.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder mainly due to gradual loss of dopaminergic neurons in the substantia nigra. Although the causative genes for autosomal recessive PD, Parkin, PINK1 and DJ-1, share a common pathway, at least in part, in mitochondrial quality control and protein quality control, their precise relationship remains elusive. Previous studies suggested the limitation of gene-modified mice model to solve this problem. DT40 is an avian leukosis virus-induced chicken B cell line with an exceptionally high ratio of targeted to random DNA integration, which enables efficient targeted disruption of multiple genes of interest. We generated DJ-1-deficient DT40 cells and analyzed PD-related phenotypes. These cells exhibited vulnerability to oxidative stress, mitochondrial dysfunction and fragmentation. Importantly, we showed that mitochondrial membrane potential and morphology are available for the phenotype analysis in DT40. These results suggest that genetically engineered DT40 cells would serve as a relevant model of PD, and help understand the genetic and functional relationship among multiple causative genes. Furthermore, in line with the recent concept of PD as a systemic disorder, elucidating the pathomechanism of PD using DT40 would lead to the development of noninvasive diagnostic tools and drug screening assays using patient-derived lymphocytes.

A case of follicular lymphoma associated with paraneoplastic cerebellar degeneration.

Paraneoplastic neurological disorders (PND) are neurological effects of malignancy that are recognized as immune-mediated disorders caused by aberrant expression of a tumor antigen that is normally expressed in the nervous system. We report a case of cerebellar ataxia which turned out to be paraneoplastic cerebellar degeneration, a subtype of PND that develops cerebellar symptoms, that was caused by follicular lymphoma. After chemotherapy, the patient attained sufficient improvement of cerebellar symptoms along with complete remission of lymphoma. Paraneoplastic cerebellar degeneration should be recognized as a rare complication of lymphoma as it is important to start proper treatment before the neurological symptoms become irreversible.