Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

The transcription factor NF-YA is crucial for neural progenitor maintenance during brain development.

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.

GPATCH4 contributes to nucleolus morphology and its dysfunction impairs cell viability.

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated ß-galactosidase (SA-ß-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability.

Modeling familial and sporadic Parkinson's disease in small fishes.

The establishment of animal models for Parkinson's disease (PD) has been challenging. Nevertheless, once established, they will serve as valuable tools for elucidating the causes and pathogenesis of PD, as well as for developing new strategies for its treatment. Following the recent discovery of a series of PD causative genes in familial cases, teleost fishes, including zebrafish and medaka, have often been used to establish genetic PD models because of their ease of breeding and gene manipulation, as well as the high conservation of gene orthologs. Some of the fish lines can recapitulate PD phenotypes, which are often more pronounced than those in rodent genetic models. In addition, a new experimental teleost fish, turquoise killifish, can be used as a sporadic PD model, because it spontaneously manifests age-dependent PD phenotypes. Several PD fish models have already made significant contributions to the discovery of novel PD pathological features, such as cytosolic leakage of mitochondrial DNA and pathogenic phosphorylation in α-synuclein. Therefore, utilizing various PD fish models with distinct degenerative phenotypes will be an effective strategy for identifying emerging facets of PD pathogenesis and therapeutic modalities.

Current trends in basic research on Parkinson's disease: from mitochondria, lysosome to α-synuclein.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and other brain regions. A key pathological feature of PD is the abnormal accumulation of α-synuclein protein within affected neurons, manifesting as Lewy bodies and Lewy neurites. Despite extensive research efforts spanning several decades, the underlying mechanisms of PD and disease-modifying therapies remain elusive. This review provides an overview of current trends in basic research on PD. Initially, it discusses the involvement of mitochondrial dysfunction in the pathogenesis of PD, followed by insights into the role of lysosomal dysfunction and disruptions in the vesicular transport system. Additionally, it delves into the pathological and physiological roles of α-synuclein, a crucial protein associated with PD pathophysiology. Overall, the purpose of this review is to comprehend the current state of elucidating the intricate mechanisms underlying PD and to outline future directions in understanding this disease.

Fish Models for Exploring Mitochondrial Dysfunction Affecting Neurodegenerative Disorders.

Neurodegenerative disorders are characterized by the progressive loss of neuronal structure or function, resulting in memory loss and movement disorders. Although the detailed pathogenic mechanism has not been elucidated, it is thought to be related to the loss of mitochondrial function in the process of aging. Animal models that mimic the pathology of a disease are essential for understanding human diseases. In recent years, small fish have become ideal vertebrate models for human disease due to their high genetic and histological homology to humans, ease of in vivo imaging, and ease of genetic manipulation. In this review, we first outline the impact of mitochondrial dysfunction on the progression of neurodegenerative diseases. Then, we highlight the advantages of small fish as model organisms, and present examples of previous studies regarding mitochondria-related neuronal disorders. Lastly, we discuss the applicability of the turquoise killifish, a unique model for aging research, as a model for neurodegenerative diseases. Small fish models are expected to advance our understanding of the mitochondrial function in vivo, the pathogenesis of neurodegenerative diseases, and be important tools for developing therapies to treat diseases.

Evaluation of Ectopic Mitochondrial DNA in HeLa Cells.

The presence of ectopic DNA in the cytoplasm induces inflammation and cell death. It has been widely reported that leakage of nuclear DNA into the cytoplasm can mainly be sensed by cyclic GMP-AMP synthase (cGAS). We recently reported that mitochondria-derived cytoplasmic double-stranded DNA (dsDNA) that has escaped lysosomal degradation induces significant cytotoxicity in cultured cells and in vivo. Cytoplasmic mitochondrial DNA is assumed to be involved in various diseases and disorders, and more and more papers have been published confirming this. On the other hand, the current method for evaluating mitochondrial DNA in the cytoplasm may not be quantitative. Here, we introduce in detail a method to evaluate ectopic mitochondrial DNA in cells. This method is useful in basic research as well as in the study of aging, Parkinson's disease, Alzheimer's disease, heart failure, autoimmune diseases, cancer, and other conditions.

Zebrafish, Medaka and Turquoise Killifish for Understanding Human Neurodegenerative/Neurodevelopmental Disorders.

In recent years, small fishes such as zebrafish and medaka have been widely recognized as model animals. They have high homology in genetics and tissue structure with humans and unique features that mammalian model animals do not have, such as transparency of embryos and larvae, a small body size and ease of experiments, including genetic manipulation. Zebrafish and medaka have been used extensively in the field of neurology, especially to unveil the mechanisms of neurodegenerative diseases such as Parkinson's and Alzheimer's disease, and recently, these fishes have also been utilized to understand neurodevelopmental disorders such as autism spectrum disorder. The turquoise killifish has emerged as a new and unique model animal, especially for ageing research due to its unique life cycle, and this fish also seems to be useful for age-related neurological diseases. These small fishes are excellent animal models for the analysis of human neurological disorders and are expected to play increasing roles in this field. Here, we introduce various applications of these model fishes to improve our understanding of human neurological disorders.

Modelling Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) Using Mice and Zebrafish.

Autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD) are two debilitating neurodevelopmental disorders. The former is associated with social impairments whereas the latter is associated with inattentiveness, hyperactivity, and impulsivity. There is recent evidence that both disorders are somehow related and that genes may play a large role in these disorders. Despite mounting human and animal research, the neurological pathways underlying ASD and ADHD are still not well understood. Scientists investigate neurodevelopmental disorders by using animal models that have high similarities in genetics and behaviours with humans. Mice have been utilized in neuroscience research as an excellent animal model for a long time; however, the zebrafish has attracted much attention recently, with an increasingly large number of studies using this model. In this review, we first discuss ASD and ADHD aetiology from a general point of view to their characteristics and treatments. We also compare mice and zebrafish for their similarities and discuss their advantages and limitations in neuroscience. Finally, we summarize the most recent and existing research on zebrafish and mouse models of ASD and ADHD. We believe that this review will serve as a unique document providing interesting information to date about these models, thus facilitating research on ASD and ADHD.

Esophageal High-Resolution Manometry for Diagnosing the Severity of the Chronic Intestinal Pseudo-Obstruction: A Case Series.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a severe and refractory intestinal motility disorder. However, due to its rarity and difficult histological investigation, its pathophysiology has not been characterized. AIM: Therefore, in this study, we aimed to determine the role of esophageal high-resolution manometry (HRM) in CIPO and the histological and clinical characteristics of the disease. METHODS: Patients with CIPO were analyzed for clinical characteristics; histological findings; and clinical courses after therapeutic intervention. In addition, HRM was performed to determine the esophageal involvement. RESULTS: Eleven patients were diagnosed with CIPO, and five required the long period of parenteral nutrition showing impaired esophageal motility including achalasia and absent contractility diagnosed with HRM. The four of these five cases showed acute onset of the CIPO following the triggering events of pregnancy, appendicitis, and surgery. In contrast, other six patients with normal or Jackhammer esophagus on HRM had moderate severity of CIPO with gradual onset. The histological analyses revealed that the loss of the intestinal neural ganglion cells and layers by inflammation, destruction, and atrophy are related to the severity of the clinical course of the disease and esophageal HRM findings of achalasia and absent contractility. CONCLUSIONS: HRM may be useful to diagnose the severity of the clinical course and to determine the therapeutic options for CIPO.

Parkinson's disease pathogenesis from the viewpoint of small fish models.

Parkinson's disease is a neurodegenerative disorder that involves movement discloses, degeneration of dopaminergic neurons, and presence of cytoplasmic inclusion bodies. Various animal models have been developed and small fish including zebrafish and medaka fish have recently been employed as a new model for Parkinson disease. In this review, we summarize fish models of Parkinson's disease mainly using our own findings and explain two major hypotheses of PD: lysosome dysfunction theory and mitochondrial dysfunction theory. Finally, we discuss the potential for future application of small fish model.

Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

Dopamine system, cerebellum, and nucleus ruber in fish and mammals.

Small teleost fish including zebrafish and medaka have been used as animal models for research because of their small body size, vast amounts of eggs produced, their rapid development, low husbandry costs, and transparency during embryogenesis. Although the body size and appearance seem different, fish and mammals including human still possess anatomical and functional similarities in their brains. This review summarizes the similarities of brain structures and functions between teleost fish and mammalian brains, focusing on the dopamine system, functional regionalization of the cerebellum, and presence of the nucleus ruber.

Cerebrospinal fluid injection into adult zebrafish for disease research.

A modified method of cerebrospinal fluid injection was developed for the efficient and reliable administration of substances to the zebrafish central nervous system. The accuracy of this modified method was evaluated using Alexa Fluor dye injection. A high survival ratio was achieved due to the simplicity of the procedure and ice-tricaine combined anaesthesia. To validate this new method, we injected ammonium chloride, which successfully blocked lysosome function resulting in elevated LC3-II and the accumulation of ubiquitinated proteins. Injection of human α-synuclein fibrils initiated a prion-like propagation of α-synuclein pathology in zebrafish. This method can be used to investigate the effects of various substances and the propagation of α-synuclein in the central nervous system.

Functional regionalization of the teleost cerebellum analyzed in vivo.

There has been accumulating evidence for a regionalized organization of the cerebellum, which was mostly deduced from anatomical mapping of axonal projections of cerebellar afferents. A likewise regionalization of the cerebellar output has been suggested from lesion studies and dye-tracer experiments, but its physiological targets as well as the functional relevance of such an output regionalization are less clear. Ideally, such functional regionalization should be proven noninvasively in vivo. We here provide evidence for such a regionalization of the output from the cerebellar cortex by genetically encoded transneuronal mapping of efferent circuits of zebrafish Purkinje neurons. These identified circuits correspond to distinct regionalized Purkinje cell activity patterns in freely behaving zebrafish larvae during the performance of cerebellar-dependent behaviors. Furthermore, optogenetic interrogation of selected Purkinje cell regions during animal behavior confirms the functional regionalization of Purkinje cell efferents and reveals their contribution to behavior control as well as their function in controlling lateralized behavioral output. Our findings reveal how brain compartments serve to fulfill a multitude of functions by dedicating specialized efferent circuits to distinct behavioral tasks.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

PINK1 and Parkin complementarily protect dopaminergic neurons in vertebrates.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective dopaminergic cell loss in the substantia nigra, but its pathogenesis remains unclear. The recessively inherited familial PD genes PARK2 and PARK6 have been attributed to mutations in the Parkin and PTEN-induced kinase 1 (PINK1) genes, respectively. Recent reports suggest that PINK1 works upstream of Parkin in the same pathway to regulate mitochondrial dynamics and/or conduct autophagic clearance of damaged mitochondria. This phenomenon is preserved from Drosophila to human cell lines but has not been demonstrated in a vertebrate animal model in vivo. Here, we developed a medaka fish (Oryzias latipes) model that is deficient in Pink1 and Parkin. We found that despite the lack of a conspicuous phenotype in single mutants for Pink1 or Parkin, medaka that are deficient in both genes developed phenotypes similar to that of human PD: late-onset locomotor dysfunction, a decrease in dopamine levels and a selective degeneration of dopaminergic neurons. Further analysis also revealed defects in mitochondrial enzymatic activity as well as cell death. Consistently, PINK1 and Parkin double-deficient MEF showed a further decrease in mitochondrial membrane potential and mitochondrial complex I activity as well as apoptosis compared with single-deficient MEF. Interestingly, these mitochondrial abnormalities in Parkin-deficient MEF were compensated by exogenous PINK1, but not by disease-related mutants. These results suggest that PINK1 and Parkin work in a complementary way to protect dopaminergic neurons by maintaining mitochondrial function in vertebrates.

[Difficult Airway Management of a Patient with the Ossification of Anterior Longitudinal Ligament].

We report a case of difficult airway management (DAM) with the ossification of anterior longitudinal ligament (OALL). A 66-year-old man complained of pharyngeal discomfort. He was diagnosed with OALL, and planned to have a surgery under general anesthesia. We expected DAM due to the limitation of cervical mobility and airway obstruction caused by OALL. We succeeded in awake intubation with video laryngoscope and tracheal tube introducer.

TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency.

OBJECTIVE: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink(-/-) ) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency. METHODS: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation. RESULTS: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink(-/-) larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink(-/-) larvae. There was also marked microglial activation in pink(-/-) larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis. INTERPRETATION: Pink1(-/-) zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD.

Single housing of juveniles accelerates early-stage growth but extends adult lifespan in African turquoise killifish.

Within the same species, individuals exhibiting faster growth tend to have shorter lifespans, even if their fast growth arises from early-life pharmacological interventions. However, in vertebrates, the impact of the early-life environment on the growth rate and lifespan has not been fully elucidated. In this study, by utilizing the short-lived African turquoise killifish, which is suitable for a comprehensive life-stage analysis in a brief timeframe, we explored the effects of housing density during the juvenile stage on holistic life traits. As a result, we found that lower housing densities resulted in faster growth, but led to longer adult lifespan, which was contrary to the common notion. Furthermore, the single-housed adult fish displayed a longer egg-laying period than did their group-housed counterparts. Our transcriptome analysis also demonstrated that, in terms of internal transcriptional programs, the life stage progression and aging process of single-housed fish were slower than those of group-housed fish. Collectively, our results suggest that sharing housing with others in early life might influence whole-life attributes, potentially leading to specific life history traits beyond the typical relationship between the growth rate and lifespan.