Yasuo Kuroda

Activation of TRPA1 Channel Facilitates Excitatory Synaptic Transmission in Substantia Gelatinosa Neurons of the Adult Rat Spinal Cord

TRPA1 is expressed in primary sensory neurons and hair cells, and it is proposed to be activated by cold stimuli, mechanical stimuli, or pungent ingredients. However, its role in regulating synaptic transmission has never been documented yet. In the present study, we examined whether activation of the TRPA1 channels affects synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. A chief ingredient of mustard oil, allyl isothiocyanate (AITC), superfused for 2 min markedly increased the frequency and amplitude of spontaneous EPSCs (sEPSCs), which was accompanied by an inward current. Similar actions were produced by cinnamaldehyde and allicin. The AITC-induced increases in sEPSC frequency and amplitude were resistant to tetrodotoxin (TTX) and La3+, whereas being significantly reduced in extent in a Ca2+-free bath solution. In the presence of glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activities. The AITC-induced increases in sEPSC frequency and amplitude were reduced by ruthenium red, whereas being unaffected by capsazepine. AITC also increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents; this AITC action was abolished in the presence of TTX or glutamate receptor antagonists. These results indicate that TRPA1 appears to be localized not only at presynaptic terminals on SG neurons to enhance glutamate release, but also in terminals of primary afferents innervating onto spinal inhibitory interneurons, which make synapses with SG neurons. This central modulation of sensory signals may be associated with physiological and pathological pain sensations.

Autoantibodies against heterogeneous nuclear ribonucleoprotein B1 in CSF of MS patients.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play an important role as the autoantigens in certain autoimmune disorders including neurological diseases such as HTLV‐1–associated myelopathy/tropical spastic paraparesis and paraneoplastic neurological syndromes. To clarify their implication in multiple sclerosis (MS), we assayed antibodies (Abs) against hnRNP A and B proteins in sera and cerebrospinal fluid (CSF) of MS patients and compared the results with 25 patients with other neurological diseases (ONDs). Using recombinant hnRNP A1, A2, and B1 proteins and Western blotting for the assay, we found Abs against hnRNP B1 in CSF from 32 of 35 MS patients (91.4%) but not in any sera or CSF of the 25 OND patients. Most notably, no Abs against hnRNP B1 were found in sera of all 22 MS patients examined. Although Abs against hnRNP A1 and A2 were concomitantly found in CSF reacting with B1, their incidence and immunoreactivity were lower or weaker than those of anti–hnRNP B1 Abs. There was no correlation between the reactivity of CSF with hnRNP B1 and CSF parameters—such as the number of the cells and the IgG level—or clinical parameters—such as duration of illness and disease activity. The selective generation of Abs against hnRNP B1 in CSF was shown to be highly specific for MS, which makes them a disease marker. Ann Neurol 2004

Clinical Characteristics by Topographical Distribution of Brain Microbleeds, With a Particular Emphasis on Diffuse Microbleeds

From the perspective of the underlying pathogenesis of primary intracerebral hemorrhage (pICH), the topographical distribution of brain microbleeds (MBs) is divided into the lobar area and the deep brain or infratentorial areas. We investigated clinical features, including ambulatory blood pressure (ABP), of patients with MBs distributed in both areas (diffuse MBs). A total of 124 patients with first-ever acute stroke were enrolled prospectively. Gradient-echo T2∗-weighted magnetic resonance imaging (MRI) was performed using a 1.5-T scanner. Patients were classified into 4 groups: MBs-negative group (n=68), those with MBs in lobar areas (lobar group; n=6), those with MBs in deep or infratentorial areas (deep or infratentorial group; n=31), and those with MBs in both areas (diffuse group; n=19). The admission casual BP (CBP) was recorded in all patients, and ABP was measured in the ischemic stroke patients. There were significant differences in the distribution of MBs (P=.004) among the 6 stroke subtypes. All stroke subtypes except transient ischemic attack had diffuse MBs; pICH had the highest prevalence of it (35%). The severity of white matter hyperintensity (WMH) differed among the 4 groups (P < .0001), with the diffuse group having the highest prevalence of early confluent (47%) and confluent types (21%). ABP and CBP were significantly higher in the deep and diffuse groups compared with the MBs-negative group, but did not differ between the lobar group and the MBs-negative group. Our data suggest that diffuse MBs are associated with hypertensive stroke, elevated BP, and severe WMH. The pathogenesis of diffuse MBs may be related to the more severe microangiopathy involved in hypertensive arteriopathy and cerebral amyloid angiopathy.

Significantly increased antibody response to heterogeneous nuclear ribonucleoproteins in cerebrospinal fluid of multiple sclerosis patients but not in patients with human T-lymphotropic virus type I–associated myelopathy/tropical spastic paraparesis

It has been reported that antibodies (Abs) against heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with human T-lymphotropic virus type I (HTLV-I)—associated myelopathy/tropical spastic paraparesis (HAM/TSP) and multiple sclerosis (MS). However, these studies were done under nonmasked conditions. In order to determine whether Abs against hnRNPs associate with HAM/TSP and MS, the authors assayed Abs against two major hnRNPs, hnRNP A1 and A2/B1, in 105 cerebrospinal fluid (CSF) samples under fully masked conditions. Samples included 40 cases of HAM/TSP, 28 of MS, and 37 of other neurological diseases. Anti-hnRNP A1 Abs, and especially anti-hnRNP A2/B1 Abs, were found significantly more often in the CSF of MS patients than in other groups. However, there was no difference in the incidence of anti-hnRNP A1 Abs between HAM/TSP and other disease groups.

α Pix enhances mutant huntingtin aggregation

Huntingtons disease is caused by polyglutamine-expanded mutant huntingtin (muhtt), an aggregation-prone protein. We identified the Pak-interacting exchange factor (alpha Pix/Cool2) as a novel huntingtin (htt) interacting protein, after screening actin-cytoskeleton organization-related factors. Using immunoprecipitation experiments, we show that alpha Pix binds to both the N-terminal of wild-type htt (wthtt) and mutant htt (muthtt). Colocalization studies revealed that alpha Pix accumulates in muthtt aggregates. Deletion analysis suggested that the dbl homology (DH) and pleckstrin homology (PH) domains of alpha Pix are required for its interaction with htt. Overexpression of alpha Pix enhanced muthtt aggregation by inducing SDS-soluble muthtt-muthtt interactions. Conversely, knocking down alpha Pix attenuated muhtt aggregation. These findings suggest that alpha Pix plays an important role in muthtt aggregation.

Identification of a new homozygous frameshift insertion mutation in the SIL1 gene in 3 Japanese patients with Marinesco-Sjögren syndrome.

Marinesco-Sjögren syndrome (MSS) is an autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts, progressive muscular weakness, and developmental and mental retardation. Recently, mutations in the SIL1 gene on chromosome 5q31 have been shown to be a cause of MSS. We sequenced the entire SIL1-coding region in 3 unrelated Japanese patients with classical MSS and identified a novel homozygous frameshift insertion mutation, 936_937insG, in exon 9 in all 3 patients.

Cloning and functional characterization of a rat Daxx that functions as a corepressor for the androgen receptor.

The androgen receptor (AR), a ligand‐activated nuclear transcription factor, plays an important role in endocrine system development and homeostasis. AR‐mediated gene transcription is modulated by a wide variety of coregulator proteins, but corepressors of AR have not been well characterized. We have isolated a rat homologue of Daxx, a possible AR repressor. The clone obtained comprised a 2196 bp open reading frame, corresponding to a sequence of 731 amino acids with 80.5% homology to mouse. Basal transcription of the isolated clone was repressed in transient transcription experiments using a Gal4 reporter gene assay. Furthermore, a mammalian two‐hybrid assay showed a strong interaction between Daxx and promyelocytic leukemia (PML) protein. These results indicate that the isolated clone is a rat homologue of Daxx. The rat Daxx (rDaxx) interacted with the androgen receptor (AR): in co‐immunoprecipitation experiments, AR formed complexes with anti‐Daxx antibody. Furthermore, overexpression of Daxx suppressed AR‐mediated transcriptional activity in HeLa and CV1 cells in transient transfection experiments. Taken together, our results suggest that Daxx may function as a corepressor for the androgen receptor.

Four mutations of the spastin gene in Japanese families with spastic paraplegia

AbstractHereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower limbs. HSP is caused by failure of development or selective degeneration of the corticospinal tracts, which contain the longest axons in humans. The most common form of HSP is caused by mutations of the spastin gene (SPAST), located on chromosome 2p21-p22, which encodes spastin, one of the ATPases associated with diverse cellular activities (AAA). In this study, we detected four causative mutations of SPAST among 14 unrelated patients with spastic paraplegia. Two missense mutations (1447A→G, 1207C→G) and two deletion mutations (1465delT, 1475-1476delAA) were located in the AAA cassette region. Three of these four mutations were novel. Previous reports and our results suggest that the frequency of SPAST mutations is higher among Japanese patients with autosomal dominant HSP, although SPAST mutations are also observed in patients with sporadic spastic paraplegia.