Masayoshi Tada

Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3′-phosphate and 3′-phosphoglycolate ends

Aprataxin is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia/ataxia with oculomotor apraxia type 1 (EAOH/AOA1), the clinical symptoms of which are predominantly neurological. Although aprataxin has been suggested to be related to DNA single-strand break repair (SSBR), the physiological function of aprataxin remains to be elucidated. DNA single-strand breaks (SSBs) continually produced by endogenous reactive oxygen species or exogenous genotoxic agents, typically possess damaged 3′-ends including 3′-phosphate, 3′-phosphoglycolate, or 3′-α, β-unsaturated aldehyde ends. These damaged 3′-ends should be restored to 3′-hydroxyl ends for subsequent repair processes. Here we demonstrate by in vitro assay that recombinant human aprataxin specifically removes 3′-phosphoglycolate and 3′-phosphate ends at DNA 3′-ends, but not 3′-α, β-unsaturated aldehyde ends, and can act with DNA polymerase β and DNA ligase III to repair SSBs with these damaged 3′-ends. Furthermore, disease-associated mutant forms of aprataxin lack this removal activity. The findings indicate that aprataxin has an important role in SSBR, that is, it removes blocking molecules from 3′-ends, and that the accumulation of unrepaired SSBs with damaged 3′-ends underlies the pathogenesis of EAOH/AOA1. The findings will provide new insight into the mechanism underlying degeneration and DNA repair in neurons.

Long-term therapeutic efficacy and safety of low-dose tacrolimus (FK506) for myasthenia gravis

OBJECTIVE To elucidate the long-term therapeutic efficacy and safety of low-dose FK506 (tacrolimus) in patients with myasthenia gravis (MG). PATIENTS AND METHODS We treated nine patients with MG (all women: age range: 35-83 years (mean: 51.1 years); MGFA classification: 4 type IIa, 4 type IIb, and 1 type IVb patients) with FK506 for more than 24 months (observation period: 24-46 months). All the patients had undergone extended thymectomy before FK506 treatment; two patients (22.2%) had noninvasive thymoma and six (66.7%) had thymic hyperplasia. We evaluated total Quantitative MG (Q-MG) score, anti-acetylcholine receptor (AChR) antibody titer in the blood, interleukin 2 (IL-2) production in peripheral blood mononuclear cells (PBMCs), administration dosage of prednisolone (PSL), and adverse effects of FK506. RESULTS A reduction in steroid dosage of 50% without worsening of the symptoms was observed 1 year after FK506 administration in three out of six steroid-dependent MG patients (50.0%). The total Q-MG scores (range: 0-39 points) at 6 months and 1 year after FK506 administration improved by 3 points or more in six (66.7%) and seven (77.8%) out of nine patients, respectively. The efficacy of FK506 was maintained for more than 2 years. Although adverse effects were observed in three patients (33.3%), these were not serious. CONCLUSIONS Our study indicates that low-dose FK506 treatment may be efficacious not only in controlling intractable myasthenic symptoms, but also in reducing steroid dosage, and that FK506 is safe as an adjunctive drug to PSL for MG treatment for a maximum of 3 years.

Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation

The clinical characteristics of colony stimulating factor 1 receptor (CSF1R) related adult‐onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) have been only partially elucidated.

Coexistence of Huntington’s disease and amyotrophic lateral sclerosis: a clinicopathologic study

We report a retrospective case series of four patients with genetically confirmed Huntington’s disease (HD) and sporadic amyotrophic lateral sclerosis (ALS), examining the brain and spinal cord in two cases. Neuropathological assessment included a polyglutamine recruitment method to detect sites of active polyglutamine aggregation, and biochemical and immunohistochemical assessment of TDP-43 pathology. The clinical sequence of HD and ALS varied, with the onset of ALS occurring after the mid-50’s in all cases. Neuropathologic features of HD and ALS coexisted in both cases examined pathologically: neuronal loss and gliosis in the neostriatum and upper and lower motor neurons, with Bunina bodies and ubiquitin-immunoreactive skein-like inclusions in remaining lower motor neurons. One case showed relatively early HD pathology while the other was advanced. Expanded polyglutamine-immunoreactive inclusions and TDP-43-immunoreactive inclusions were widespread in many regions of the CNS, including the motor cortex and spinal anterior horn. Although these two different proteinaceous inclusions coexisted in a small number of neurons, the two proteins did not co-localize within inclusions. The regional distribution of TDP-43-immunoreactive inclusions in the cerebral cortex partly overlapped with that of expanded polyglutamine-immunoreactive inclusions. In the one case examined by TDP-43 immunoblotting, similar TDP-43 isoforms were observed as in ALS. Our findings suggest the possibility that a rare subset of older HD patients is prone to develop features of ALS with an atypical TDP-43 distribution that resembles that of aggregated mutant huntingtin. Age-dependent neuronal dysfunction induced by mutant polyglutamine protein expression may contribute to later-life development of TDP-43 associated motor neuron disease in a small subset of patients with HD.

Roles of inositol 1,4,5-trisphosphate receptors in spinocerebellar ataxias.

Modulation of the intracellular calcium concentration is a ubiquitous signaling system involved in the control of numerous biological processes in a wide variety of cells. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which act as calcium release channels in the ER membrane, play a key role in the regulation of intracellular calcium concentration. IP3R type 1 (IP3R1) is the major neuronal IP3R isoform in the central nervous system and particularly abundant in cerebellar Purkinje cells. Heterozygous deletions or missense mutations in ITPR1, which encodes IP3R1, result in autosomal dominantly inherited spinocerebellar ataxias (SCAs), including SCA types 15 (SCA15) and 29 (SCA29). In addition, homozygous missense mutations in carbonic anhydrase-related protein VIII (CARP), which suppresses the ability of IP3 to bind to IP3R1, cause a recessively inherited ataxia with mild cognitive impairment with/without quadrupedal gait. Moreover, cytosolic calcium overload with excessive IP3R1 activity has been implicated in the pathogenesis of other SCAs, including SCA types 2 (SCA2) and 3 (SCA3). These facts indicate that dysregulation of IP3R-mediated calcium signaling is linked to the pathogenesis of SCAs. Here, we focus on the molecular basis of SCA15 and SCA29, which are caused by mutations in ITPR1. In addition, we discuss other SCAs whose pathogenesis may be linked to aberrant activation of IP3R-mediated Ca(2+) signaling.

Genotype–phenotype correlations in early onset ataxia with ocular motor apraxia and hypoalbuminaemia

Early onset ataxia with ocular motor apraxia and hypoalbuminaemia/ataxia-oculomotor apraxia 1 is a recessively inherited ataxia caused by mutations in the aprataxin gene. We previously reported that patients with frameshift mutations exhibit a more severe phenotype than those with missense mutations. However, reports on genotype-phenotype correlation in early onset ataxia with ocular motor apraxia and hypoalbuminaemia are controversial. To clarify this issue, we studied 58 patients from 39 Japanese families, including 40 patients homozygous for c.689_690insT and nine patients homozygous or compound heterozygous for p.Pro206Leu or p.Val263Gly mutations who were compared with regard to clinical phenotype. We performed Kaplan-Meier analysis and log-rank tests for the ages of onset of gait disturbance and the inability to walk without assistance. The cumulative rate of gait disturbance was lower among patients with p.Pro206Leu or p.Val263Gly mutations than among those homozygous for the c.689_690insT mutation (P=0.001). The cumulative rate of inability to walk without assistance was higher in patients homozygous for the c.689_690insT mutation than in those with p.Pro206Leu or p.Val263Gly mutations (P=0.004). Using a Cox proportional hazards model, we found that the homozygous c.689_690insT mutation was associated with an increased risk for onset of gait disturbance (adjusted hazard ratio: 6.60) and for the inability to walk without assistance (adjusted hazard ratio: 2.99). All patients homozygous for the c.689_690insT mutation presented ocular motor apraxia at <15 years of age. Approximately half the patients homozygous for the c.689_690insT mutation developed cognitive impairment. In contrast, in the patients with p.Pro206Leu or p.Val263Gly mutations, only ∼50% of the patients exhibited ocular motor apraxia and they never developed cognitive impairment. The stepwise multivariate regression analysis using sex, age and the number of c.689_690insT alleles as independent variables revealed that the number of c.689_690insT alleles was independently and negatively correlated with median motor nerve conduction velocities, ulnar motor nerve conduction velocities and values of serum albumin. In the patient with c.[689_690insT]+[840delT], p.[Pro206Leu]+[Pro206Leu] and p.[Pro206Leu]+[Val263Gly] mutations, aprataxin proteins were not detected by an antibody to the N-terminus of aprataxin. Furthermore Pro206Leu and Val263Gly aprataxin proteins are unstable. However, the amount of the 689_690insT aprataxin messenger RNA was also decreased, resulting in more dramatic reduction in the amount of aprataxin protein from the c.689_690insT allele. In conclusion, patients with early onset ataxia with ocular motor apraxia and hypoalbuminaemia homozygous for the c.689_690insT mutation show a more severe phenotype than those with a p.Pro206Leu or p.Val263Gly mutation.

Redefining cerebellar ataxia in degenerative ataxias: lessons from recent research on cerebellar systems

Recent advances in our understanding of neurophysiological functions in the cerebellar system have revealed that each region involved in degenerative ataxias contributes differently. To regulate voluntary movements, the cerebellum forms internal models within its neural circuits that mimic the behaviour of the sensorimotor system and objects in the external environment. The cerebellum forms two different internal models: forward and inverse. The forward model is formed by efference copy signals conveyed by the corticopontocerebellar system, and it derives the estimated consequences for action. The inverse model describes sequences of motor commands to accomplish an aim. During motor learning, we improve internal models by comparing the estimated consequence of an action from the forward model with the actual consequence of the action produced by the inverse model. The functions of the cerebellum encompass the formation, storage and selection of internal models. Considering the neurophysiological properties of the cerebellar system, we have classified degenerative ataxias into four types depending on which system is involved: Purkinje cells, the corticopontocerebellar system, the spinocerebellar system and the cerebellar deep nuclei. With regard to their respective contributions to the internal models, we speculate that loss of Purkinje cells leads to malformation of the internal models, whereas disturbance of the afferent system, corticopontocerebellar system or spinocerebellar system leads to mis-selection of the proper internal model. An understanding of the pathophysiological properties of ataxias in each degenerative ataxia enables the development of new methods to evaluate ataxias.

Transportin 1 accumulates in FUS inclusions in adult-onset ALS without FUS mutation.

Accumulation of a protein, DNA/RNA binding protein fused in sarcoma (FUS), as cytoplasmic inclusions in neurones and glial cells in the central nervous system (CNS) is the pathological hallmark of amyotrophic lateral sclerosis (ALS) with FUS mutations (ALS-FUS) [1,2] as well as certain subtypes of frontotemporal lobar degeneration (FTLD-FUS) [3,4], the latter being unassociated with FUS mutations. While the inclusions in ALS-FUS contain only FUS, those in FTLD-FUS show co-accumulation of three proteins of the FET protein family, i.e. in addition to FUS, Ewing’s sarcoma (EWS) and TATA-binding proteinassociated factor 15 (TAF15) [5]. These findings strongly suggest that a more complex derangement of transportinmediated nuclear import of proteins accounts for the disease process in FTLD-FUS in comparison to ALS-FUS. Recently, Neumann et al. reported that the inclusions in FTLD-FUS subtypes were strongly labelled for transportin 1 (TRN1) and that, as expected, the inclusions in ALS-FUS were completely unreactive for this protein [6]. Here we report an adult patient who exhibited a clinically pure ALS phenotype without FUS mutations (ALS-FUS) and cytoplasmic inclusions showing coaccumulation of FET and TRN1 proteins, confirming that ALS-FUS and FTLD-FUS represent part of a spectrum of FUS proteinopathy without FUS mutation. A 65-year-old Japanese woman became aware of muscle weakness in her hands. Three years later, she was diagnosed as having ALS. Thereafter, bulbar palsy and respiratory distress progressed gradually; at the age of 70 years, tracheotomy and respirator support became necessary. The patient died of gastrointestinal bleeding at the age of 74 years, about 9 years after disease onset. There was no family history of neurological disorders, including ALS. During the disease course, dementia was not evident. We sequenced and found no mutations in the all coding regions of the FUS gene. The brain was small and weighed 820 g (brainstem and cerebellum, 110 g) before fixation. The spinal cord showed marked atrophy. Histologically, loss of myelinated fibres was observed in the spinal white matter except for the posterior columns; the lateral corticospinal tracts appeared to be only mildly affected (Figure 1a). Neuronal loss and gliosis were also evident in the spinal anterior horns (Figure 1b) and brainstem hypoglossal nucleus. The presence of slightly basophilic round inclusions in the remaining lower motor neurones was a feature (Figure 1c). No Bunina bodies were found. In the cerebral cortex, mild neuronal loss was noted in the motor cortex. Immunostaining with an antibody against FUS (polyclonal, SigmaAldrich, St Louis, USA; 1:50) revealed widely distributed positive neuronal cytoplasmic inclusions (NCIs) in the CNS, including the spinal anterior horn and motor cortex (Figure 1d,e). The distribution and severity of FUS lesions and neuronal loss are shown inTable 1. FUS immunostaining also revealed positive glial cytoplasmic inclusions (GCIs) (Figure 1f). These cytoplasmic inclusions were also labelled by anti-ubiquitin (polyclonal, Dako, Glostrup, Denmark; 1:800) (Figure 1g) and anti-p62 (monoclonal; BD Bioscience, San Jose, CA, USA; 1:1000) antibodies (Figure 1h). No neuronal nuclear inclusions (NIIs) were found.The results of immunostaining for a-internexin and TDP-43 were all negative (data not shown). Immunostaining with an antibody against transportin 1 (TRN1) (monoclonal, Abcam, Cambridge, UK; 1:200) also revealed clearly positive NCIs (Figure 2a) and GCIs. Such inclusions were also labelled with anti-TAF15 (polyclonal, Bethyl Lab, Montgomery, USA; 1:200) (Figure 2b) and anti-EWS (monoclonal, Santa Cruz, Santa Cruz, USA; 1:200) antibodies (Figure 2c). TRN1 and FUS were sometimes fully or partially colocalized in the same neurones (Figure 2d–f,g–i) and glial cells. The ratio of the colocalization (TRN1/FUS) in NCIs was about 50%. FTLD-FUS can be classified into three pathological subtypes, atypical FTLD-U, neuronal intermediate filament inclusion disease (NIFID), and basophilic inclusion body disease (BIBD) on the basis of the morphology and distribution pattern of FUS-positive NCIs and NIIs [3]. Atypical FTLD-U is characterized by compact, round to oval kidneyshaped NCIs and vermiform NIIs in the neocortex, granule cells of the dentate gyrus, striatum and some other brain regions. NIFID is characterized by FUS-positive NCIs and NIIs as well as less predominant type IV interfilament-, a-internexinand neurofilament-positive NCIs. Lastly,

Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy

Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12. Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2, indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion.This study identifies TTTCA- and TTTTA-repeat expansions in benign adult familial myoclonic epilepsy. Cortical neurons from affected people exhibit RNA foci containing these expanded repeats, suggesting RNA toxicity as the mechanism underlying disease pathogenesis.

Characteristic microglial features in patients with hereditary diffuse leukoencephalopathy with spheroids.

To clarify the histopathological alterations of microglia in the brains of patients with hereditary diffuse leukoencephalopathy with spheroids (HDLS) caused by mutations of the gene encoding the colony stimulating factor‐1 receptor (CSF‐1R).