Hiroto Eguchi

TDP-43 mutation in familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. Accumulating evidence has shown that 43kDa TAR‐DNA–binding protein (TDP‐43) is the disease protein in ALS and frontotemporal lobar degeneration. We previously reported a familial ALS with Bumina bodies and TDP‐43‐positive skein‐like inclusions in the lower motor neurons; these findings are indistinguishable from those of sporadic ALS. In three affected individuals in two generations of one family, we found a single base‐pair change from A to G at position 1028 in TDP‐43, which resulted in a Gln‐to‐Arg substitution at position 343. Our findings provide a new insight into the molecular pathogenesis of ALS. Ann Neurol 2008;63:538–542

TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation

Recently, 43-kDa TAR DNA-binding protein (TDP-43) was identified as a component of ubiquitinated inclusions (UIs) in sporadic amyotrophic lateral sclerosis (SALS). To clarify whether TDP-43 immunoreactivity is present in neuronal inclusions in familial ALS (FALS), we examined immunohistochemically the brains and spinal cords from four cases of FALS, two with Cu/Zn superoxide dismutase (SOD1) gene mutation and two without, together with three cases of SALS and three control subjects, using two antibodies, one polyclonal and one monoclonal, against TDP-43. Neuropathologically, the SOD1-related FALS cases were characterized by Lewy body-like hyaline inclusions (LBHIs) in the lower motor neurons. On the other hand, the SOD1-unrelated FALS cases showed degeneration restricted to the upper and lower motor neuron systems, with Bunina bodies (BBs) and UIs in the lower motor neurons, being indistinguishable from SALS. No cytoplasmic TDP-43 immunoreactivity was observed in the control subjects or SOD1-related FALS cases; LBHIs were ubiquitinated, but negative for TDP-43. UIs observed in the SALS and SOD1-unrelated FALS cases were clearly positive for TDP-43. BBs were negative for this protein. Interestingly, in these SALS and FALS cases, glial cells were also found to have cytoplasmic TDP-43-positive inclusions. These findings indicate that the histological and molecular pathology of SALS can occur as a phenotype of FALS without SOD1 mutation.

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

Summary1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF.2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimers type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase.3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients.4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.

Japanese amyotrophic lateral sclerosis patients with GGGGCC hexanucleotide repeat expansion in C9ORF72

Background A GGGGCC hexanucleotide repeat expansion in C9ORF72 occurs on a chromosome 9p21 locus that is linked with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in white populations. The diseases resulting from this expansion are referred to as ‘c9FTD/ALS’. It has been suggested that c9FTD/ALS arose from a single founder. However, the existence of c9FTD/ALS in non-white populations has not been evaluated. Results We found two index familial ALS (FALS) patients with c9FTD/ALS in the Japanese population. The frequency of c9FTD/ALS was 3.4% (2/58 cases) in FALS. No patients with sporadic ALS (n=110) or control individuals (n=180) had the expansion. Neuropathological findings of an autopsy case were indistinguishable from those of white patients. Although the frequency of risk alleles identified in white subjects is low in Japanese, one patient had all 20 risk alleles and the other had all but one. The estimated haplotype indicated that the repeat expansion in these patients was located on the chromosome with the risk haplotype identified in white subjects. Conclusions C9ORF72 repeat expansions were present in a Japanese cohort of ALS patients, but they were rare. Intriguingly, Japanese patients appear to carry the same risk haplotype identified in white populations.

Total Tau Protein in Cerebrospinal Fluid and Diffusion-Weighted MRI as an Early Diagnostic Marker for Creutzfeldt-Jakob Disease

Background: We have recently begun to doubt the effectiveness of periodic sharp wave complexes observed on electroencephalographs and the detection of 14-3-3 protein in cerebrospinal fluid (CSF) as diagnostic criteria for Creutzfeldt-Jakob disease (CJD). Diffusion-weighted magnetic resonance imaging (DWI) and the detection of total tau (t-tau) protein in CSF may be more sensitive diagnostic criteria. Methods: Among 44 CJD patients, we selected 21 subjects that suffered from early-stage CJD, which was defined as cases in the 6 weeks following the onset of the disease. The sensitivities of DWI and electroencephalographs, as well as those of t-tau protein, 14-3-3 protein, neuron-specific enolase (NSE), and S-100b protein in CSF were compared as diagnostic markers for early-stage CJD. Results: NSE, S-100b protein, t-tau protein, and 14-3-3 protein were detected in the samples from 57.1, 4.8, 95.2, and 76.2% of the 21 early-stage CJD patients, respectively. Additionally, DWI was used to positively identify 90.5% of these cases. Conclusion: We concluded that t-tau protein was the most sensitive of the diagnostic markers for CJD. Moreover, the data in this study showed that detection of t-tau protein combined with DWI identified 98% of the early-stage cases, and these tests should be included as diagnostic criteria for CJD.

Acetazolamide acts directly on the human skeletal muscle chloride channel

Acetazolamide, a carbonic anhydrase inhibitor, is used empirically in neuromuscular diseases with episodic ataxia, weakness, and myotonia, although not all of the mechanisms responsible for its therapeutic effects are understood. To elucidate whether acetazolamide acts directly on the human skeletal muscle voltage‐gated chloride channel (ClC‐1), which is associated with myotonia, we evaluated the effects of acetazolamide on ClC‐1 expressed in cultured mammalian cells, using whole‐cell recording. Acetazolamide significantly shifted the voltage dependency of the open probability (Po) toward negative potentials in a dose‐dependent manner, resulting in an increase of chloride conductance at voltages near the resting membrane potential. This effect was attenuated when using a pipette solution containing 30 mmol/L Hepes. These results suggest that acetazolamide can influence the voltage‐dependent opening gate of ClC‐1 through a mechanism related to intracellular acidification by inhibiting carbonic anhydrase, and that the therapeutic effects of acetazolamide in neuromuscular diseases may be mediated by activation of ClC‐1. Muscle Nerve, 2006

Large deletion involving the 5'-UTR in the spastin gene caused mild phenotype of autosomal dominant hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) due to mutations in the spastin gene (SPG4) located to 2p22‐p21 is the most common form of autosomal dominant (AD) HSP. We performed PCR‐based direct sequencing of SPG4, followed by a linkage analysis and subsequent Southern blot analysis in large Japanese kindred where 20 of 33 members were evaluated neurologically, and consequently 6 were affected with HSP. Clinical evaluation showed that the mean age at disease onset of the patients was older and the disability was less severe than those of previously reported typical patients with SPG4 mutations. Direct sequencing of genomic DNA and RT‐PCR product did not show a SPG4 mutation despite of a strong linkage to the SPG4 locus at 2p. Southern blot analysis suggested a deletion involving the 5′‐UTR of SPG4. Further sequence analysis confirmed a heterozygous 2307‐bp deletion spanning from the 5′‐UTR to intron 1 of SPG4. The results suggested that transcription of the mutated allele starts from an authentic initiation site, but lacks an authentic translational start site of exon 1 because of a deficient splice donor site and coding region. The abnormal transcripts may result in rapid RNA decay. The novel refractory mutation we identified widens the spectrum of SPG4 mutations. These findings suggest that structural genomic abnormalities of SPG4 are more frequent than expected, and this explains previously reported cases more feasibly in which SPG4 mutations were failed to be identified but the disease was linked to 2p.

A CLCN1 mutation in dominant myotonia congenita impairs the increment of chloride conductance during repetitive depolarization

Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.

Chronological changes in MRI and CSF biochemical markers in Creutzfeldt-Jakob disease patients.

Background: There are currently no markers for evaluating chronological changes in Creutzfeldt-Jakob disease (CJD). We examined if chronological changes in biochemical markers in cerebrospinal fluid (CSF) and diffusion-weighted magnetic resonance imaging (DWI) were utilizable for this purpose. Methods: Ten independent patients were divided into two groups of 5 patients each. We analyzed CSF biochemical markers, DWI and the clinical course in one group. In the remaining group, only the CSF biochemical markers were analyzed before and after the onset of akinetic mutism. Results: The level of total tau (t-tau) protein in CSF in the early phase after disease onset was 2,655 ± 423.9 pg/ml, reaching a mean peak of 14,675 ± 1,240 pg/ml in the middle phase and gradually declining after that. Just before patients deteriorated into akinetic mutism, t-tau protein titers reached a maximum (8,786 ± 2,975 pg/ml). There were dramatic changes in t-tau protein levels throughout the clinical course, unlike the other markers. DWI was not always utilizable, because of discordance with clinical symptoms seen in this study. Four cases exhibited peaks in t-tau protein levels while the patients fell into akinetic mutism except 1 case. Conclusion: Our results suggest that t-tau protein is the most sensitive marker of disease progression in CJD patients.

C9ORF72 repeat-associated non-ATG-translated polypeptides are distributed independently of TDP-43 in a Japanese patient with c9ALS.

Hexanucleotide (GGGGCC) repeat expansion in a noncoding region of C9ORF72 is the major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS) in the Caucasian population [1], but it is very rare in the Japanese population, possibly because of the difference in genetic background [2,3]. TDP-43 pathology indistinguishable from that of non-mutational ALS/FTLD-TDP has been observed in c9FTD/ALS [4]. In addition, the presence of p62-, ubiquitinand ubiquilin-positive, and TDP-43-negative inclusions in the cerebellar cortex and hippocampus has been reported to be a unique and consistent feature in Caucasian patients with c9FTD/ALS [5,6]. Recently, it was demonstrated that these TDP-43-negative inclusions are derived from aggregated dipeptide repeat (DPR) proteins bidirectionally translated from the expanded repeat in C9ORF72 by repeat associated non-ATG (RAN) translation, and that such DPR protein pathology is widely distributed in the central nervous system (CNS) [7,8]. However, it still remains unknown whether these distinct neuropathological features are also reproduced in Japanese patients with c9FTD/ALS. In the present study, we performed an immunohistochemical analysis focusing especially on DPR proteins in a Japanese patient with C9ORF72 repeat expansion (c9ALS) [2], who to our knowledge represents the only autopsy case of this genetic disease to have been reported in the Japanese population so far. The present study was conducted with approval from the Institutional Review Board of Niigata University. The clinical and pathological findings in this case have been reported previously (case 4 [9]). Briefly, the patient had a sibling who had also been diagnosed as having ALS. At the age of 61, he noticed hand clumsiness, and progressively developed bulbar palsy and limb weakness. He died 20 months after disease onset due to respiratory failure. He had no clinical features suggestive of dementia. The presence of C9ORF72 repeat expansion was confirmed by repeat-primed PCR using the frozen cerebellar tissue [2]. The accurate hexanucleotide repeat length was unknown, because we could not perform Southern blot analysis due to lack of the amount of genomic DNA. Histologically, neuronal loss and gliosis were evident in the spinal anterior horns, brainstem motor nuclei and motor cortex, as well as degeneration in the anterior and lateral columns of the spinal cord. Bunina bodies and ubiquitin-positive skein-like inclusions were observed in the remaining lower motor neurones. Neuronal and glial (oligodendrocytic) cytoplasmic inclusions (NCIs and GCIs) recognized with anti-TDP-43 (polyclonal, Protein Tec Group, Chicago, IL, USA; 1:4000) were present in the lower motor nuclei and motor cortex, and much less frequently in the subcortical non-motor nuclei, such as the basal ganglia, whereas no such NCIs or GCIs were observed in the cerebellar cortex and hippocampus [9]. There were p62-positive and TDP43-negative NCIs in the cerebellar granule cells and in the granule cells and pyramidal CA4-CA2 neurones of the hippocampus; at that time, we failed to show phosphorylated TDP-43 (pTDP-43)-positive NCIs in the lower motor nuclei and motor cortex [2]. In the present study, we confirmed that NCIs and GCIs recognized with a ‘polyclonal’ antibody against pTDP-43 (pS409/410; Cosmo Bio, Tokyo, Japan; 1: 1000) were present with the same distribution pattern as that of TDP-43 mentioned above (Figure 1a,c). Immunostaining with an antibody against another RNA-binding protein, RNA-binding motif 45 (RBM45) (polyclonal, SigmaAldrich, St. Louis, MO, USA; 1:50), which has been known to accumulate in inclusions in ALS [10], also revealed a distribution of positive inclusions strikingly similar to that of TDP-43-positive inclusions, although such NCIs and GCIs were comparatively small in number. The characteristic morphologies of NCIs and GCIs were shared by both anti-TDP-43/anti-pTDP-43 and antiRBM45 (Figure 1a–d). We generated polyclonal antibodies against putative DPR proteins from the GGGGCC repeat by RAN translation [11]. Although immunostaining with three antibodies against different polypeptides, poly Gly-Ala (GA), poly Gly-Pro and poly Gly-Arg, arising from RAN translation revealed similar DPR protein pathology, abundant positive