Tomohiko Ishihara

Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis

Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Sporadic four-repeat tauopathy with frontotemporal lobar degeneration, Parkinsonism, and motor neuron disease: a distinct clinicopathological and biochemical disease entity

Tau is the pathological protein in several neurodegenerative disorders classified as frontotemporal lobar degeneration (FTLD), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). We report an unusual tauopathy in three Japanese patients presenting with Parkinsonism and motor neuron disease (neuroimaging revealed frontotemporal cerebral atrophy in two patients who were examined). At autopsy, all cases showed FTLD with the most severe neuronal loss and gliosis evident in the premotor and precentral gyri. Although less severe, such changes were also observed in other brain regions, including the basal ganglia and substantia nigra. In the spinal cord, loss of anterior horn cells and degeneration of the corticospinal tract were evident. In addition, the affected regions exhibited neuronal cytoplasmic inclusions resembling neurofibrillary tangles. Immunostaining using antibodies against hyperphosphorylated tau and 4-repeat tau revealed widespread occurrence of neuronal and glial cytoplasmic inclusions in the central nervous system; the astrocytic tau lesions were unique, and different in morphology from astrocytic plaques in CBD, or tufted astrocytes in PSP. However, immunoblotting of frozen brain samples available in two cases revealed predominantly 4R tau, with the approximately 37-kDa and 33-kDa low-molecular mass tau fragments characteristic of CBD and PSP, respectively. No mutations were found in the tau gene in either of the two cases. Based on these clinicopathological, biochemical, and genetic findings, we consider that the present three patients form a distinct 4R tauopathy associated with sporadic FTLD.

Alteration of POLDIP3 splicing associated with loss of function of TDP-43 in tissues affected with ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by selective loss of motor neurons. In the ALS motor neurons, TAR DNA-binding protein of 43 kDa (TDP-43) is dislocated from the nucleus to cytoplasm and forms inclusions, suggesting that loss of a nuclear function of TDP-43 may underlie the pathogenesis of ALS. TDP-43 functions in RNA metabolism include regulation of transcription, mRNA stability, and alternative splicing of pre-mRNA. However, a function of TDP-43 in tissue affected with ALS has not been elucidated. We sought to identify the molecular indicators reflecting on a TDP-43 function. Using exon array analysis, we observed a remarkable alteration of splicing in the polymerase delta interacting protein 3 (POLDIP3) as a result of the depletion of TDP-43 expression in two types of cultured cells. In the cells treated with TDP-43 siRNA, wild-type POLDIP3 (variant-1) decreased and POLDIP3 lacking exon 3 (variant-2) increased. The RNA binding ability of TDP-43 was necessary for inclusion of POLDIP3 exon 3. Moreover, we found an increment of POLDIP3 variant-2 mRNA in motor cortex, spinal cord and spinal motor neurons collected by laser capture microdissection with ALS. Our results suggest a loss of TDP-43 function in tissues affected with ALS, supporting the hypothesis that a loss of function of TDP-43 underlies the pathogenesis of ALS.

New HSN2 mutation in Japanese patient with hereditary sensory and autonomic neuropathy type 2

The authors report a Japanese patient with hereditary sensory and autonomic neuropathy type 2 (HSAN2) who has a new mutation of the HSN2 gene. The pathologic findings of the patient matched those of Canadian patients. They identified a homozygous 1134-1135 ins T mutation, resulting in a frameshift, and the subsequent premature stop codon at residue 378. These observations support the hypothesis that HSN2 is a causative gene for HSAN2.

Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder. In motor neurons of ALS, TAR DNA binding protein-43 (TDP-43), a nuclear protein encoded by TARDBP, is absent from the nucleus and forms cytoplasmic inclusions. TDP-43 auto-regulates the amount by regulating the TARDBP mRNA, which has three polyadenylation signals (PASs) and three additional alternative introns within the last exon. However, it is still unclear how the autoregulatory mechanism works and how the status of autoregulation in ALS motor neurons without nuclear TDP-43 is. Here we show that TDP-43 inhibits the selection of the most proximal PAS and induces splicing of multiple alternative introns in TARDBP mRNA to decrease the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. When TDP-43 is depleted, the TARDBP mRNA uses the most proximal PAS and is increased in the cytoplasm. Finally, we have demonstrated that in ALS motor neurons—especially neurons with mislocalized TDP-43—the amount of TARDBP mRNA is increased in the cytoplasm. Our observations indicate that nuclear TDP-43 contributes to the autoregulation and suggests that the absence of nuclear TDP-43 induces an abnormal autoregulation and increases the amount of TARDBP mRNA. The vicious cycle might accelerate the disease progression of ALS.

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.

Depression and psychiatric symptoms preceding onset of dementia in a family with early-onset Alzheimer disease with a novel PSEN1 mutation.

Sirs, PSEN1 mutations are the most common cause of autosomal dominant early-onset familial Alzheimer disease (EO-FAD) in diverse ethnic groups [1–3]. Cognitive decline is a cardinal clinical feature in patients with PSEN1 mutations; however, various accompanying clinical manifestations have been reported [4]. We report a family with a novel PSEN1 mutation who developed depression and psychiatric symptoms preceding the onset of dementia. The first patient (Patient 1, III-1 in Fig. 1a) has suffered from depression and has undergone antidepressant treatment from the age of 29. She gradually became apathetic at the age of 47. At the age of 48, she developed visual and auditory hallucinations and personality change. She was referred to us for neurological evaluation. Her cognition was impaired as determined using the revised version of the Hasegawa dementia scale (HDS-R) [5] of 11/30. Neurological examination revealed rigidity of the left upper limb and postural instability, suggesting the presence of parkinsonism. Brain MRI showed atrophy of the medial temporal lobe with right predominance. 99mTc-ECD SPECT revealed hypoperfusion in the posterior cingulate gyri as well as in the frontal and parietotemporal cortices with right hemisphere predominance. The younger sister of Patient 1 (Patient 2, III-2 in Fig. 1a) was diagnosed as having depression when she was in high school. Since then, she has experienced repeated manic and depressive bipolar episodes. Because hallucinations, abnormal cravings, and spatial disorientation were noted at the age of 40, she was admitted to a psychiatric hospital. She was examined by us at the age of 42 and found to exhibit parkinsonian gait, stooped posture, rigidity of limbs, and severe cognitive decline as determined by HDS-R (0/30). Brain MRI showed predominant medial temporal atrophy. 99mTc-ECD SPECT revealed severe hypoperfusion in the posterior cingulated gyri and bilateral parietal area. On the basis of clinical and imaging findings, we clinically suspected them as having EO-FAD, although they exhibited unusual accompanying symptoms. Their mother, uncles, and grandfather were demented according to an interview with family members (Fig. 1a). A genetic screening of mutations in APP, PSEN1, PSEN2, and MAPT was performed after obtaining written, informed consent from the patients and their caregivers. We identified a heterozygous G to C transition in exon 6 of PSEN1 in the patients, which resulted in a previously undescribed Leu173Phe mutation. This mutation was absent in 110 healthy controls from the same population, as determined by allele-specific PCR methods (Fig. 1b). Codon 173 leucine is located in the third transmembrane domain of PSEN1 and is highly conserved among species. The APOE genotype was 3/4 in both patients. To examine the pathological characteristics of the PSEN1 mutation, we established neuroblastoma-derived K. Kasuga T. Ishihara O. Onodera T. Ikeuchi (&) Department of Molecular Neuroscience, Brain Research Institute, Niigata University, 1 Asahimachi, Chuo-ku, Niigata 951-8585, Japan e-mail: ikeuchi@bri.niigata-u.ac.jp

Relapse of multiple sclerosis in a patient retaining CCR7-expressing T cells in CSF under fingolimod therapy

Fingolimod acts as a functional antagonist of the sphingosine-1-phosphate receptor, and it traps lymphocytes in secondary lymphoid organs and precludes their migration into the central nervous system. We report the case of a patient who suffered a relatively severe relapse of multiple sclerosis (MS) during the initial 3 months of fingolimod therapy, with retention of CCR7 expression on CD4+ T cells in the cerebrospinal fluid (CSF) despite decreased numbers of lymphocytes and decreased expression of CCR7 on CD4+ T cells in the blood. These data suggest that fingolimod may cause differential effects on the CSF and blood lymphocytes of patients with MS during the initial months of therapy.

Atypical micrographia associated with corticostriatal white matter lesions in systemic lupus erythematosus

Micrographia is a heterogeneous condition in which various parts of the CNS may be involved. An anatomical substrate for micrographia, however, remains to be established. Here, we report on a patient with systemic lupus erythematosus (SLE), who presented with atypical micrographia, which was associated with bilateral lesions in the corticostriatal white matter. A 30-year-old right-handed woman was diagnosed as having SLE in 2002. She had concomitant leukocytopenia, arthritis, nephritis and high titres of antinuclear antibodies, which satisfied the American Rheumatism Association criteria for SLE. Thereafter, maintenance treatment using corticosteroids was started. She was admitted to the Niigata University Medical and Dental Hospital, Niigata, Japan, in February 2005, because of a high fever and headache with affective incontinence. Examination showed her muscle strength and the sensory function of her extremities to be normal. She did not have involuntary movements or akinetic–rigid symptoms, as her gait was normal and no rigidity was observed in the neck, body and extremities. No impairment was seen in the rapid alternative movements of her hands. She was well oriented and cooperative. Aphasia was absent; her speech was well articulated and grammatically correct, and she had no difficulty in naming objects. She had no abnormalities in praxis, showing an excellent capacity in imitating and pantomiming, and in using tools with either hand. She could perform a fist–palm-alternating task swiftly. Orofacial apraxia, visuospatial disturbance, unilateral spatial …

Minor splicing pathway is not minor any more: Implications for the pathogenesis of motor neuron diseases

To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR‐DNA binding protein 43 kDa (TDP‐43) must be elucidated. TDP‐43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP‐43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate‐rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre‐mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre‐mRNA splicing. Although the major class of spliceosomes regulates most pre‐mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre‐mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.