Hodaka Yamakado

A progressive dopaminergic phenotype associated with neurotoxic conversion of α-synuclein in BAC-transgenic rats

Conversion of soluble α-synuclein into insoluble and fibrillar inclusions is a hallmark of Parkinsons disease and other synucleinopathies. Accumulating evidence points towards a relationship between its generation at nerve terminals and structural synaptic pathology. Little is known about the pathogenic impact of α-synuclein conversion and deposition at nigrostriatal dopaminergic synapses in transgenic mice, mainly owing to expression limitations of the α-synuclein construct. Here, we explore whether both the rat as a model and expression of the bacterial artificial chromosome construct consisting of human full-length wild-type α-synuclein could exert dopaminergic neuropathological effects. We found that the human promoter induced a pan-neuronal expression, matching the rodent α-synuclein expression pattern, however, with prominent C-terminally truncated fragments. Ageing promoted conversion of both full-length and C-terminally truncated α-synuclein species into insolube and proteinase K-resistant fibres, with strongest accumulation in the striatum, resembling biochemical changes seen in human Parkinsons disease. Transgenic rats develop early changes in novelty-seeking, avoidance and smell before the progressive motor deficit. Importantly, the observed pathological changes were associated with severe loss of the dopaminergic integrity, thus resembling more closely the human pathology.

PINK1 and Parkin complementarily protect dopaminergic neurons in vertebrates

Parkinsons 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.

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.

α-synuclein BAC transgenic mice as a model for Parkinson's disease manifested decreased anxiety-like behavior and hyperlocomotion.

α-Synuclein (α-syn), the main component of Lewy bodies, was identified as a genetic risk factor for idiopathic Parkinsons disease (PD). As a model for PD, we generated human α-syn bacterial artificial chromosome transgenic mice (BAC tg mice) harboring the entire human α-syn gene and its gene expression regulatory regions. The α-syn BAC tg mice manifested decreased anxiety-like behaviors which may reflect non-motor symptoms of early PD, and they exhibited increased SERT expression that may be responsible for decreased anxiety-like behaviors. Our α-syn BAC tg mice could be a valuable tool to evaluate α-syn gene dosage effects in vivo.

Chronic overload of SEPT4, a parkin substrate that aggregates in Parkinson’s disease, causes behavioral alterations but not neurodegeneration in mice

BackgroundIn autosomal recessive early-onset Parkinsonism (PARK2), the pathogenetic process from the loss of function of a ubiquitin ligase parkin to the death of dopamine neurons remains unclear. A dominant hypothesis attributes the neurotoxicity to accumulated substrates that are exempt from parkin-mediated degradation. Parkin substrates include two septins; SEPT4/CDCrel-2 which coaggregates with α-synuclein as Lewy bodies in Parkinson’s disease, and its closest homolog SEPT5/CDCrel-1/PNUTL1 whose overload with viral vector can rapidly eliminate dopamine neurons in rats. However, chronic effects of pan-neural overload of septins have never been examined in mammals. To address this, we established a line of transgenic mice that express the largest gene product SEPT454kDa via the prion promoter in the entire brain.ResultsHistological examination and biochemical quantification of SEPT4-associated proteins including α-synuclein and the dopamine transporter in the nigrostriatal dopamine neurons found no significant difference between Sept4Tg/+ and wild-type littermates. Thus, the hypothetical pathogenicity by the chronic overload of SEPT4 alone, if any, is insufficient to trigger neurodegenerative process in the mouse brain. Intriguingly, however, a systematic battery of behavioral tests revealed unexpected abnormalities in Sept4Tg/+ mice that include consistent attenuation of voluntary activities in distinct behavioral paradigms and altered social behaviors.ConclusionsTogether, these data indicate that septin dysregulations commonly found in postmortem human brains with Parkinson’s disease, schizophrenia and bipolar disorders may be responsible for a subset of behavioral abnormalities in the patients.

Idiopathic Parkinson's disease patient‐derived induced pluripotent stem cells function as midbrain dopaminergic neurons in rodent brains

Patient‐specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinsons disease (PD) patients, however, their vulnerability and the transmission of pathological α‐Synuclein are possible drawbacks that may prevent PD‐specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC‐derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α‐Synuclein transgenic mice, PDiPSC‐derived DA neurons did not cause pathological α‐Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD.

Diagnostic utility of FDG-PET in neurolymphomatosis: report of five cases

Neurolymphomatosis (NL) is a rare condition involving the infiltration of lymphoma cells into the peripheral nervous system. NL can be primary or secondary in the setting of an unknown or known hematologic malignancy, respectively. Here, we report five cases in which F-18 2-fluoro-2-deoxy-glucose positron emission tomography/computed tomography (F-18 FDG-PET/CT) had great value for diagnosing NL. Two cases were rare primary NL, and the other three were secondary NL. Clinical presentations were asymmetric sensorimotor disturbances in the extremities with or without involvement of cranial nerves. Furthermore, all patients experienced spontaneous pain in the face or affected extremities. Cerebrospinal fluid analysis was cytologically negative in two of five cases. Gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) detected abnormalities in the cranial nerves, nerve roots, and cauda equina in all cases except case 1 and the recurrent stage of case 2. F-18 FDG-PET/CT showed clear visualization of almost all the lymphomatous involvement of peripheral nerves and other tissues in all patients. Furthermore, F-18 FDG-PET/CT detected abnormalities including asymptomatic lesions that were not detected with MRI, and also identified the appropriate lesion for diagnostic biopsy. However, as in case 3, the lesions in the left oculomotor nerve and the cauda equina were detected only with Gd-enhanced MRI, which has superior spatial resolution. In conclusion, F-18 FDG-PET/CT is a sensitive modality that can suggest the presence of malignancy and identify appropriate places for diagnostic biopsies. It is especially useful when combined with Gd-enhanced MRI, even in patients with primary NL that is usually difficult to diagnose.

Exploring the Pathogenetic Mechanisms underlying Parkinson's Disease in Medaka Fish

Teleost fish have recently been employed as a model for human neurodegenerative diseases. We used toxin exposure and genetic engineering to develop models of Parkinsons disease (PD) in the teleost fish, medaka. Among the toxins examined, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors, and tunicamycin all induced important features of PD in medaka. Specifically, these agents induced dopaminergic cell loss and reduced spontaneous movement, and the latter three toxins produced inclusion bodies that were ubiquitously distributed in the medaka brain. Despite the extensive distribution of these inclusion bodies, the middle diencephalic dopaminergic neurons were particularly susceptible to the effect of the toxins, suggesting that this cluster of dopaminergic neurons is analogous to the human substantia nigra. We have also created a variety of different genetic models using the Targeting Induced Local Lesions in Genomes (TILLING) method, and found that neither PTEN-induced putative kinase 1 (PINK1) mutants nor Parkin mutants disclosed significant dopaminergic cell loss. Surprisingly however, PINK1 and Parkin double mutants exhibited selective dopamine cell loss, as well as aggregation and deficit of mitochondrial activity. Another mutant, the ATP13A2 mutant, also expressed a PD phenotype, exhibiting marked cathepsin D reduction and fingerprint-like structures that are generally found in lysosome storage diseases. Taken together, these data indicate that medaka fish can serve as a new animal model for PD. In this review, we summarize our data and discuss the potential for future application of this animal model.

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

Parkinsons 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.

Zonisamide inhibits monoamine oxidase and enhances motor performance and social activity

Zonisamide (ZNS) is an effective drug for not only motor symptoms but also non-motor symptoms in Parkinsons disease. However, the actions of ZNS as an anti-Parkinsonian drug are not well understood. To clarify the actions of ZNS in vivo, we administered ZNS to mice and examined the effects on neurotransmitter metabolism and behaviors, focusing on motor and non-motor symptoms. Administration of ZNS decreased dopamine (DA) turnover in various brain regions, including the striatum. In behavioral tests, ZNS enhanced locomotor activity and novelty seeking in the open field test, light-dark transition test, and the social interaction test. Consistent with these results of DA metabolism in ZNS-treated mice, monoamine oxidase activity was significantly inhibited by ZNS in primary neurons and astrocytes. Collectively, these data suggest that ZNS inhibits monoamine oxidase activity and decreases DA turnover, which increases locomotor activity and novelty seeking in mice. ZNS is potentially useful to improve not only motor symptoms but also neuropsychiatric non-motor symptoms such as apathy in PD.