Yoshihiro Nihei

Modeling familial Alzheimer’s disease with induced pluripotent stem cells

Alzheimers disease (AD) is the most common form of age-related dementia, characterized by progressive memory loss and cognitive disturbance. Mutations of presenilin 1 (PS1) and presenilin 2 (PS2) are causative factors for autosomal-dominant early-onset familial AD (FAD). Induced pluripotent stem cell (iPSC) technology can be used to model human disorders and provide novel opportunities to study cellular mechanisms and establish therapeutic strategies against various diseases, including neurodegenerative diseases. Here we generate iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I), and characterize the differentiation of these cells into neurons. We find that FAD-iPSC-derived differentiated neurons have increased amyloid β42 secretion, recapitulating the molecular pathogenesis of mutant presenilins. Furthermore, secretion of amyloid β42 from these neurons sharply responds to γ-secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs. Our findings demonstrate that the FAD-iPSC-derived neuron is a valid model of AD and provides an innovative strategy for the study of age-related neurodegenerative diseases.

Characterization of alternative isoforms and inclusion body of the TAR DNA-binding protein-43

TAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(−/−) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies.

Neurological response to early removal of ovarian teratoma in anti-NMDAR encephalitis

We report an 18-year-old woman with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, who developed psychiatric symptoms, progressive unresponsiveness, dyskinesias, hypoventilation, hypersalivation and seizures. Early removal of an ovarian teratoma followed by plasma exchange and corticosteroids resulted in a prompt neurological response and eventual full recovery. Serial analysis of antibodies to NR1/NR2B heteromers of the NMDAR showed an early decrease of serum titres, although the cerebrospinal fluid titres correlated better with clinical outcome. The patients’ antibodies reacted with areas of the tumour that contained NMDAR-expressing tissue. Search for and removal of a teratoma should be promptly considered after the diagnosis of anti-NMDAR encephalitis.

Establishment of induced pluripotent stem cells from centenarians for neurodegenerative disease research

Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimers disease and familial Parkinsons disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.

Enhanced aggregation of androgen receptor in induced pluripotent stem cell-derived neurons from spinal and bulbar muscular atrophy

Background: Induced pluripotent stem cells (iPSCs) are a novel technology for modeling neurodegeneration. Results: We established spinal and bulbar muscular atrophy (SBMA)-derived iPSCs and confirmed motor neuron differentiation. Aggregation of androgen receptor in SBMA-iPSC-derived neurons is enhanced by DHT and inhibited by 17-AAG. Conclusion: SBMA-iPSCs show disease-specific biochemical features. Significance: Using SBMA-iPSCs is an innovative strategy for studying polyglutamine diseases. Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

Camptocormia in Japanese patients with Parkinson's disease: a multicenter study.

The aim of this work was to investigate the prevalence of camptocormia and the clinical characteristics of patients with camptocormia in a large population of PD patients.

Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

Background: The pathological mechanism of the potent modifier of TDP-43 toxicity, ataxin-2, is unknown. Result: Ataxin-2 modified the subcellular distributions of truncated TDP-43 and mutant FUS. Conclusion: Increased ataxin-2 leads to a mislocation of TDP-43 and FUS, leading the RNA dysregulation. Significance: An aberrant distribution of TDP-43 and FUS mediated by ataxin-2 may be a key therapeutic target against ALS. The RNA-binding proteins TDP-43 and Fused in Sarcoma (FUS) play central roles in neurodegeneration associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Both proteins are components of messenger ribonucleoprotein (mRNP) granules and show cytoplasmic mislocalization in affected tissues. Recently, ataxin-2 was identified as a potent modifier of TDP-43 toxicity in an RNA-dependent manner. This study investigated to clarify how ataxin-2 modifies the TDP-43 and FUS pathological pathway. The expression of cytoplasmic TDP-43, the 35-kDa C-terminal fragment (TDP-p35f), and mutant FUS recruited ataxin-2 to mRNP granules, whereas increased ataxin-2 inhibited the mRNP granule formation of the 35-kDa C-terminal fragment and mutant FUS. A subcellular compartment analysis showed that the overexpressed ataxin-2 increased the cytoplasmic concentrations of both proteins, whereas it decreased their nuclear distributions. These data indicate that increased ataxin-2 impairs the assembly of TDP-43 and FUS into mRNP granules, leading to an aberrant distribution of RNA-binding proteins. Consequently, these sequences may exacerbate the impairment of the RNA-quality control system mediated by amyotrophic lateral sclerosis/frontotemporal lobar degeneration-associated RNA-binding proteins, which forms the core of the degenerative cascade.

Degeneration of mesencephalic dopaminergic neurons in klotho mouse related to vitamin D exposure.

Parkinsons disease (PD), which is characterized by degeneration of mesencephalic dopaminergic neurons of unclear etiology, is primarily an age-related neurodegenerative disorder, while the normal process of aging is also known to decrease the number of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). However, no consensus exists regarding how advancing age may predispose the dopaminergic system to PD. The Klotho-insufficient (klotho) mouse exhibits a syndrome that resembles human aging. Recent studies have revealed that abnormal activation of vitamin D is the major cause of this phenotype. In this study, we examined mesencephalic dopaminergic neurons of klotho mice and identified tyrosine hydroxylase-positive neurons in the SNc and VTA, and found that levels of striatal dopamine were significantly decreased with aging in klotho mice. Notably, these phenotypes were rescued by vitamin D restriction, suggesting that abnormal activation of vitamin D due to Klotho insufficiency leads to degeneration of the dopaminergic system. The present study provides new insights into the pathology of age-related degeneration of dopaminergic neurons possibly related to Klotho-mediated regulation of vitamin D.

Evidence of a link between ubiquilin 2 and optineurin in amyotrophic lateral sclerosis

A mutation in the ubiquilin 2 gene (UBQLN2) was recently identified as a cause of X-linked amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) and a major component of the inclusion bodies commonly found with a wide variety of ALS. ALS-linked mutations in UBQLN2 are clustered in a unique proline-X-X repeat region, reportedly leading to impairment of the ubiquitin proteasome system. However, the molecular properties of mutant UBQLN2 remain unclear. To gain insight into the pathogenesis of UBQLN2-linked ALS/FTD, we examined the biochemical and cellular characteristics of mutant UBQLN2 in vitro. UBQLN2 localized in Rab11-positive endosomal vesicles formed by the ALS-linked molecule optineurin (OPTN). These vesicles were ubiquitin- and p62-immunopositive and also co-localized with an initiator of the autophagic process, ULK1, after amino acid starvation. An ALS-linked mutation (E478G) in OPTN abolished vesicle formation. ALS-linked mutations in UBQLN2 additively enhanced UBQLN2 aggregation and formation of inclusion bodies, resulting in mislocation from OPTN vesicles. UBQLN2 was found to be a potent regulator of the levels of the FTD-linked secretory factor progranulin, possibly via the endosomal system, and ALS-linked mutations disturbed these functional consequences. This study demonstrates that ALS-linked mutations in both OPTN and UBQLN2 interfere with the constitution of specific endosomal vesicles, suggesting that the vesicles are involved in protein homeostasis and that these proteins function in common pathological processes. These data suggest a novel disease spectrum and provide new pathological insights into OPTN and UBQLN2, enhancing our understanding of the molecular basis of ALS/FTD.

Screening for Impaired Cognitive Domains in a Large Parkinson's Disease Population and Its Application to the Diagnostic Procedure for Parkinson's Disease Dementia

Background: Dementia is a new focus of research on improved treatment for Parkinsons disease (PD). In 2007, a screening tool for PD dementia (PD-D) was developed by the Movement Disorder Society (Level I testing), which still requires verification by a large population study. Methods: We conducted a cross-sectional and multicenter study including 13 institutions administering the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) to 304 PD patients (mean age: 70.6 ± 8.3 years; mean Hoehn and Yahr stage: 2.7 ± 0.7). Results: In all, 34.5% of the patients had MMSE scores <26; 94.3% of these patients had impairments in ≥2 cognitive domains and met the criteria for probable PD-D by Level I testing. Executive dysfunction combined with attention and memory impairment was most common (51.4%). In the Level I subtests of executive function, the score for phonemic fluency declined by <50% in patients with high MoCA scores (24-30 points) and lacked specificity for PD-D. No patient had visuospatial impairment (measured by the pentagon copying subtest) alone, and the score for pentagon copying stayed at ≥70% even in patients with low MMSE scores (12-25 points), therefore lacking sensitivity for PD-D. Conclusions: Level I testing with administration of the MMSE and MoCA is a practical and efficient screening tool for PD-D. However, the phonemic fluency and pentagon copying tests should be replaced by more specific/sensitive ones when screening for PD-D.