松浦 徹

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in COL1A1 or COL1A2. We identified a dominant missense mutation, c.3235G > A in COL1A1 exon 45 predicting p.G1079S, in a Japanese family with mild OI. As mutations in exon 45 exhibit mild to lethal phenotypes, we tested if disruption of an exonic splicing cis-element determines the clinical phenotype, but detected no such mutations. In the Japanese family, juvenile-onset hyperuricemia cosegregated with OI, but not in the previously reported Italian and Canadian families with c.3235G > A. After confirming lack of a founder haplotype in three families, we analyzed PRPSAP1 and PRPSAP2 as candidate genes for hyperuricemia on chr 17 where COL1A1 is located, but found no mutation. We next resequenced the whole exomes of two siblings in the Japanese family and identified variable numbers of previously reported hyperuricemia-associated SNPs in ABCG2 and SLC22A12. The same SNPs, however, were also detected in normouricemic individuals in three families. We then identified two missense SNVs in ZPBP2 and GPATCH8 on chromosome 17 that cosegregated with hyperuricemia in the Japanese family. ZPBP2 p.T69I was at the non-conserved region and was predicted to be benign by in silico analysis, whereas GPATCH8 p.A979P was at a highly conserved region and was predicted to be deleterious, which made p.A979P a conceivable candidate for juvenile-onset hyperuricemia. GPATCH8 is only 5.8 Mbp distant from COL1A1 and encodes a protein harboring an RNA-processing domain and a zinc finger domain, but the molecular functions have not been elucidated to date.

Reactive oxygen species and their detrimental effects on the brain after transient ischemia have been implicated in the pathogenesis of the ischemic injury. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system is currently recognized as the major cellular defense mechanism under oxidative stress, but the involvement of the Keap1-Nrf2 system in the ischemic brain injuries has not been fully investigated to date. In the present study, we investigated temporal changes of Keap1, Nrf2, and their downstream antioxidative proteins in post-ischemic mice brains with respect to spacial differences between the peri-infarct regions and the regions destined to infarct. In the peri-infarct regions, a steady level of Keap1 showed a decremental expression started at 2 h of reperfusion after 60 min of transient middle cerebral artery occlusion (tMCAO). In contrast, Nrf2 began to show a significant increase at 2 h with a peak at 8 h of reperfusion after tMCAO. Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glial cells. In the same peri-infarct region, downstream antioxidative proteins such as thioredoxin, glutathione, and heme oxygenase-1 showed significant increases at later time-points of 24-72 h of reperfusion after tMCAO. In the regions destined to infarct, a similar trend of expression changes to those in the peri-infarct regions was observed in Keap1, Nrf2, and 3 downstream antioxidative proteins with much less reactions. The changes found in this study suggest that the induced antioxidative stress proteins after cerebral ischemia may play an important endogenous neuroprotective response under oxidative stress after ischemic stroke. © 2010 Elsevier B.V.

We examined the neuroprotective effects amlodipine and/or atorvastatin in metabolic syndrome (MetS) Zucker fatty rats against transient (90 min) middle cerebral artery occlusion (MCAO). The rats were pretreated with vehicle, amlodipine, atorvastatin, or amlodipine plus atorvastatin for 28 days, and 24 hr after transient MCAO the infarct size was assessed via hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and microtubule-associated protein 1 light chain 3 (LC3) expression were examined by immunohistochemistry to evaluate apoptosis and autophagy, respectively. Compared with the vehicle group, rats treated with amlodipine or atorvastatin alone showed a significant decrease in infarct volume (P < 0.01), which was further decreased in the amlodipine plus atorvastatin group (P < 0.001). Compared with the vehicle group, the numbers of TUNEL- and LC3-positive cells were markedly reduced by amlodipine or atorvastatin alone (P < 0.01) and further decreased by amlodipine plus atorvastatin (P < 0.001). The number of apoptotic TUNEL/autophagic LC3 double-positive cells was also significantly decreased with amlodipine or atorvastatin alone compared with vehicle (P < 0.01) and was further decreased by amlodipine plus atorvastatin (P < 0.001). These data suggest additive neuroprotective effects of combination amlodipine and atorvastatin treatment after acute ischemic stroke in MetS model Zucker rats. These effects are mediated, at least in part, via antiapoptotic and antiautophagic mechanisms. Further studies are now needed to expand these preliminary results to understand fully the mechanisms involved in the protective effects of amlodipine and atorvastatin against ischemic stroke. (C) 2011 Wiley-Liss, Inc.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease, which is characterized by progressive degeneration of spinal and bulbar innervating motor neurons. However, the underlying mechanisms of motor neuron death remain poorly understood. Several candidate disease biomarkers have been detected in cerebrospinal fluid of ALS patients. The present study analyzed various cerebral spinal fluid gas parameters in ALS patients and compared these values to controls, as well as patients with cervical spondylosis, Parkinson syndrome, and spinocerebellar degeneration. Cerebral spinal fluid pH positively correlated with the ALS functional rating scale in total and limb-type ALS patients. In addition, cerebral spinal fluid pH positively correlated with shorter disease duration (less than 22 weeks). These results suggested that cerebral spinal fluid pH provides a biomarker for ALS and could reflect mechanisms of disease progression in ALS patients. (C) 2011 Elsevier B.V. All rights reserved.

Ischemic stroke is a major, urgent neurologic disorder in which reactive oxygen species (ROS) are deeply involved in the detrimental effects. Platinum nanoparticle (nPt) species are a novel and strong scavenger of such ROS, so we examined the clinical and neuroprotective effects of nPts in mouse ischemic brain. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. Upon reperfusion, nPt or vehicle was administered intravenously. At 48 hr after the tMCAO, motor function, infarct volume, immunohistochemistry of neurovascular components (endothelial NAGO, tight junctional occludin, and basal laminal collagen IV), and zymography for MMP-9 activity were examined. Superoxide anion generation at 2 hr after tMCAO was determined with oxidized hydroethidine. Compared with vehicle, treatment with nPts significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex. Immunohistochemical analyses revealed that tMCAO resulted in a minimal decrease of NAGO and occludin but a great decrease of collagen IV and a remarkable increase of MMP-9. Treatment with nPts greatly reduced this decrease of collagen IV and activation of MMP-9 and, with large reductions of MMP-9 activation on zymography and superoxide production. The present study demonstrates that treatment with nPts ameliorates the neurological scores with a large reduction in infarct size as well as the preservation of outer components of the neurovascular unit (collagen IV) and inactivation of MMP-9. A strong reduction of superoxide anion production by nPts could account for such remarkable neurobehavioral and neuroprotective effects on ischemic stroke. (C) 2011 Wiley-Liss, Inc.

Autosomal-dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. In this study, we performed genetic analysis of a unique form of SCA (SCA36) that is accompanied by motor neuron involvement. Genome-wide linkage analysis and subsequent fine mapping for three unrelated Japanese families in a cohort of SCA cases, in whom molecular diagnosis had never been performed, mapped the disease locus to the region of a 1.8 Mb stretch (LOD score of 4.60) on 20p13 (D20S906-D20S193) harboring 37 genes with definitive open reading frames. We sequenced 33 of these and observed a large expansion of an intronic GGCCTG hexanucleotide repeat in NOP56 and an unregistered missense variant (Phe265Leu) in C20orf194, but we found no mutations in PDYN and TGM6. The expansion showed complete segregation with the SCA phenotype in family studies, whereas Phe265Leu in C20orf194 did not. Screening of the expansions in the SCA cohort cases revealed four additional occurrences, but none were revealed in the cohort of 27 Alzheimer disease cases, 154 amyotrophic lateral sclerosis cases, or 300 controls. In total, nine unrelated cases were found in 251 cohort SCA patients (3.6%). A founder haplotype was confirmed in these cases. RNA foci formation was detected in lymphoblastoid cells from affected subjects by fluorescence in situ hybridization. Double staining and gel-shift assay showed that (GGCCUG)n binds the RNA-binding protein SRSF2 but that (CUG)(6) does not. In addition, transcription of MIR1292, a neighboring miRNA, was significantly decreased in lymphoblastoid cells of SCA patients. Our finding suggests that SCA36 is caused by hexanucleotide repeat expansions through RNA gain of function.

Recent reports suggest that functional or structural defect of vascular components are implicated in amyotrophic lateral sclerosis (ALS) pathology. In the present study, we examined a possible change of the neurovascular unit consisting of endothelium (PCAM-1), tight junction (occludin), and basement membrane (collagen IV) in relation to a possible activation of MMP-9 in ALS patients and ALS model mice. We found that the damage in the neurovascular unit was more prominent in the outer side and preferentially in the anterior horn of ALS model mice. This damage occurred prior to motor neuron degeneration and was accompanied by MMP-9 up-regulation. We also found the dissociation between the PCAM-1-positive endothelium and GFAP-positive astrocyte foot processes in both humans and the animal model of ALS. The present results indicate that perivascular damage precedes the sequential changes of the disease, which are held in common between humans and the animal model of ALS, suggesting that the neurovascular unit is a potential target for therapeutic intervention in ALS. (C) 2011 Wiley-Liss, Inc.

Objective: To investigate whether acoustic impairment can be one of the characteristic extracerebellar symptoms in sporadic and hereditary ataxias including spinocerebellar ataxia type 31 (SCA31). Methods: We investigated genotypes of dominant ataxia families, and determined a frequency of each form in our cohort of 154 families. Acoustic function in the groups of various forms of ataxia with multiple system atrophy of cerebellar predominance (MSA-C), cortical cerebellar atrophy (CCA), and hereditary ataxias including SCA31 was evaluated by using audiogram and brainstem auditory evoked potentials (BAEPs). Results: Genetic analysis of dominant ataxia families revealed that a frequency of SCA31 in our cohort was fewer than that reported from other areas of Japan, indicating that SCA31 is not widely distributed throughout Japan. Results of audiogram showed no significant difference of hearing levels among ataxic groups, and those of BAEPs did not support inner ear dysfunction in SCA31 in which hearing loss had initially been suggested as one of its characteristic symptoms. Conclusion: This study suggests that acoustic impairment is neither specific to SCA31, MSA-C and CCA nor useful in making a differential diagnosis among them.

Ischemic stroke is a major neurologic disorder and a leading cause of disability and death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat. The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28days, and physical and serum parameters were analyzed, then 90min of transient middle cerebral artery occlusion (tMCAO), was performed followed by immunohistochemical analyses at 24h. Without affecting serum levels of lipids, adiponectin, and leptin, the combination therapy of AM plus AT ameliorated the post-ischemic brain weight increase. The single treatment with AM or AT itself exerted neuroprotective effects with reducing inductions of MMP-9 and AT2R, as well as with preserving collagen IV, and the combination therapy of AM plus AT showed a further synergistic benefit against acute ischemic neural damages. Single AT was more protective on these 3 molecules than single AM at this time point of 24h after tMCAO. Thus, the combination therapy with AM plus AT extended the neuroprotectives effect of single treatment with AM or AT on a part of neurovascular unit and a hypertension-related receptor.

In addition to simply reducing the serum level of cholesterol, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have various pleiotrophic effects such as reducing oxidative stress, neuroinflammation, and neurotoxicity. However, such a pleiotrophic effect has not been fully studied in a new statin (pitavastatin). We examined and compared the effects of two strong statins (atorvastatin, 30 mg/kg/day, p.o. pitavastatin, 3 mg/kg/day, p.o.) on the serum level of lipids, cognitive dysfunction, senile plaque (SP) and phosphorylated tau-positive dystrophic neuritis (pτDN) in amyloid precursor protein (APP) transgenic (Tg) mice from 5 months (M) of age to 20 M. These two statins improved behavioral memory and reduced the numbers of SP and pτDN at 15 and 20 M without affecting serum lipid levels, but preserved mice brain weight in pitavastatin group at 20 M. These protective effects of statins took 10 M from the beginning of treatment to show an improvement in the present model mice, and sensitivity to the statin treatment was linked to behavioral memory, SP and pτDN in this order. These findings suggest that early treatment with both atorvastatin and pitavastatin prevented subsequent worsening of cognitive function and the amyloidogenic process, probably due to pleiotrophic effects, suggesting a therapeutic potential for Alzheimer's disease (AD). © 2010 Elsevier B.V. All rights reserved.

Stroke is a major neurologic disorder and a leading cause of death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28 days, and at 24h of tMCAO, infarct volume and immunohistochemical analyses were performed. The combination of AM plus AT treatment decreased the infarct volume stronger than each single treatment with AM or AT. The numbers of positive cells of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosin (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and advanced end glycation products (AGE) and inflammation markers such as tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1(MCP-1) decreased dramatically in the combination-treated group compared with single AM- or AT-treated group. The present study showed that single AM or AT treatment showed neuroprotective effects both with antioxidative and anti-inflammatory mechanisms, but combination therapy of AM plus AT presented a further synergistic benefit in acute ischemic neural damages.

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.

Apoptosis is one of the mechanisms contributing to neuronal degeneration in ischemic stroke. In vivo imaging of annexin V (A5) was performed at 12 hr, 24 hr, 48 hr, and 4 days after 90-min transient middle cerebral artery occlusion (tMCAO) in mice with a fluorescent protein Cy5.5. Immunohistochemistry for heat shock protein 70 (HSP70), A5, and TUNEL were also performed with brain sections after the tMCAO. In vivo fluorescence was strongly observed at 48 hr over the head, especially with removal of both head skin and skull bone. Zonal ex vivo fluorescent signals were surrounding the ischemic core, and double-positive cells with Cy5.5/exogenous A5 antibody were found in this area. HSP70 was observed at the peak time of 24 hr; A5 became detectable at 12 hr, with increasing numbers until 48 hr. The number of TUNEL-positive cells increased at 24 hr and retained the high level until 4 days, showing a dissociating temporal pattern with A5. Double-positive cells for A5/TUNEL reached their peak at 48 hr. All the data suggest that some cells still have a chance to be rescued for a long period after acute cerebral ischemia. The in vivo Cy5.5 fluorescence representing A5 signal spatially surrounding the ischemic core temporally detects an early-stage apoptosis after cerebral ischemia.

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated niTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to p-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways. (C) 2010 Wiley-Liss, Inc.

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5x10(5)) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled. Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1487-1493; doi: 10.1038/jcbfm.2010.32; published online 10 March 2010

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.

Amyotrophic lateral sclerosis is a progressive and fatal disease caused by selective death of motor neurons, and a number of these patients carry mutations in the superoxide dismutase 1 (SOD1) gene involved in ameliorating oxidative stress. Recent studies indicate that oxidative stress and disruption of mitochondrial homeostasis is a common mechanism for motor neuron degeneration in amyotrophic lateral sclerosis and the loss of midbrain dopamine neurons in Parkinson's disease. Therefore, the present study investigated the presence and alterations of familial Parkinson's disease-related proteins, PINK1 and DJ-1, in spinal motor neurons of G93ASOD1 transgenic mouse model of amyotrophic lateral sclerosis. Following onset of disease, PINK1 and DJ-1 protein expression increased in the spinal motor neurons. The activated form of p53 also increased and translocated to the nuclei of spinal motor neurons, followed by increased expression of p53-activated gene 608 (PAG608). This is the first report demonstrating that increased expression of PAG608 correlates with activation of phosphorylated p53 in spinal motor neurons of an amyotrophic lateral sclerosis model. These results provide further evidence of the profound correlations between spinal motor neurons of amyotrophic lateral sclerosis and parkinsonism-related proteins. (C) 2010 Wiley-Liss, Inc.

Spinocerebellar ataxia type 10 is caused by ATTCT repeat expansion in the ATXN10 gene in humans. We studied the evolutionary history of the human genome to determine the time and mechanism of the acquisition of unstable ATTCT repeats in the genome. We found that long interspersed element-1 (LINE-1) was inserted into ATXN10 intron 9; Alu was then inserted in the middle of LINE-1; and endogenous retrovilcus K was lastly retrotransposed in the middle of Alu. The ATTCT repeat was located on the boundary between the 3'-end of the Alu element and the direct repeat arising from LINE-1. We determined nucleotide sequences of the orthologous region of 50 individuals representing 33 primate species and compared them with the human sequence. The analysis revealed that the ATTCT repeat is present only in human and apes. Old World monkeys also possess pentanucleotide repeats, but their motifs are TGTCT and GGTCT. New World monkeys and prosimians are not informative because they lack the corresponding region in ATXN10 intron 9. Our studies dictate two parsimonious scenarios of evolution. First, a TT (C) under barT motif arose from a TTTTT motif at the junction of Alu and LINE-1, which was followed by introduction of A to make an (A) under bar TTCT motif in horminoids. Second, an ATT (C) under barT motif wits directly generated from an ancestral ATM motif in the common ancestor of catarrhines. We also demonstrate that orangutan uniquely introduced G to make a (G) under bar TTCT motif and later C to make a GTTC (C) under bar motif, where newly introduced nucleotides are underlined. Our Studies reveal that nucleotide substitutions in a poly(A) tail of the Alu element and the following amplification of pentanucleotides occurred in the lineages of Old World monkeys and hominoids and that unstable ATTCT pentanucleotide repeats originated in the common ancestor of hominoids. These findings also highlight a new aspect of the role of retrotransposons in human disease and evolution, which might be useful in investigating the mystery of human uniqueness.

Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5-3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)(n) stretch. In controls, shorter (1.5-2.0 kb) insertions lacking (TGGAA)(n) were found only rarely. The SCA31 repeat insertion's length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)(n) in vitro. Because (TGGAA)(n) is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with "inserted" microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.

Nucleosome packaging influences many aspects of DNA metabolism such as replication, repair and transcription, and via this link likely has further downstream effects on genome stability. The instability and expansion of repetitive sequences is associated with at least 42 human diseases yet the molecular conditions, contributing to repeat instability have remained largely undetermined. Previously we showed strong nucleosome formation on CAG repeats associated with spinocerebellar ataxia type 1 and very weak formation on CGG repeats associated with fragile X syndrome, and that interruption of these repeat tracts made the DNA behave more like random sequences. In this study, we determined nucleosome formation on pure and interrupted ATTCT pentanucleotides associated with spinocerebellar ataxia type 10 (SCA10). We report strong nucleosome formation on ATTCT repeats, like CAG tracts. Surprisingly, in contrast to the effect of interruptions on other repeat sequences, interruptions in the expanded ATTCT tracts further strengthened assembly with hyperacetylated histones under physiological conditions with NAP-1. These differences may contribute to phenotypic variation seen between families having pure and interrupted SCA10 repeats as well, as the overall genetic instability at the SCA10 locus. (c) 2008 Elsevier B.V. All rights reserved.

We recently reported that the intronic splice-site mutation IVS3-8G > A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor alpha subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional alpha subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G > A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3' end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions -232 and -74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil.

In humans and great apes, CHRNA1 encoding the muscle nicotinic acetylcholine receptor alpha subunit carries an inframe exon P3A, the inclusion of which yields a nonfunctional alpha subunit. In muscle, the P3A(-) and P3A(+) transcripts are generated in a 1:1 ratio but the functional significance and regulation of the alternative splicing remain elusive. An intronic mutation (IVS3-8G > A), identified in a patient with congenital myasthenic syndrome, disrupts an intronic splicing silencer (ISS) and results in exclusive inclusion of the downstream P3A exon. We found that the ISS-binding splicing trans-factor was heterogeneous nuclear ribonucleoprotein (hnRNP) H and the mutation attenuated the affinity of hnRNP for the ISS similar to 100-fold. We next showed that direct placement of hnRNP H to the 3' end of intron 3 silences, and siRNA-mediated downregulation of hnRNP H enhances recognition of exon P3A. Analysis of the human genome suggested that the hnRNPH-binding UGGG motif is overrepresented close to the 3' ends of introns. Pursuing this clue, we showed that alternative exons of GRIP1, FAS, VPS13C and NRCAM are downregulated by hnRNP H. Our findings imply that the presence of the hnRNP H-binding motif close to the 3' end of an intron is an essential but underestimated splicing regulator of the downstream exon.

Yeast carries a strictly conserved branch point sequence (BPS) of UACUA (A) under barC, whereas the human BPS is degenerative and is less well characterized. The human consensus BPS has never been extensively explored in vitro to date. Here, we sequenced 367 clones of lariat RT-PCR products arising from 52 introns of 20 human housekeeping genes. Among the 367 clones, a misincorporated nucleotide at the branch point was observed in 181 clones, for which we can precisely pinpoint the branch point. The branch points were comprised of 92.3 A, 3.3 C, 1.7 G and 2.8 U. Our analysis revealed that the human consensus BPS is simply yUn (A) under bary, where the underlined is the branch point at position zero and the lowercase pyrimidines ('y') are not as well conserved as the uppercase U and A. We found that the branch points are located 2134 nucleotides upstream of the 3' end of an intron in 83 clones. We also found that the polypyrimidine tract spans 424 nucleotides downstream of the branch point. Our analysis demonstrates that the human BPSs are more degenerative than we have expected and that the human BPSs are likely to be recognized in combination with the polypyrimidine tract and/or the other splicing cis-elements.

Trinucleotide repeat expansions are dynamic mutations causing many neurological disorders, and their instability is influenced by multiple factors. Repeat configuration seems particularly important, and pure repeats are thought to be more unstable than interrupted repeats. But direct evidence is still lacking. Here, we presented strong support for this hypothesis from our studies on spinocerebellar ataxia type 17 (SCA17). SCA17 is a typical polyglutamine disease caused by CAG repeat expansion in TBP (TATA binding protein), and is unique in that the pure expanded polyglutamine tract is coded by either a simple configuration with long stretches of pure CAGs or a complex configuration containing CAA interruptions. By small pool PCR (SP-PCR) analysis of blood DNA from SCA17 patients of distinct racial backgrounds, we quantitatively assessed the instability of these two types of expanded alleles coding similar length of polyglutamine expansion. Mutation frequency in patients harboring pure CAG repeats is 2-3 folds of those with CAA interruptions. Interestingly, the pure CAG repeats showed both expansion and deletion while the interrupted repeats exhibited mostly deletion at a significantly lower frequency. These data strongly suggest that repeat configuration is a critical determinant for instability, and CAA interruptions might serve as a limiting element for further expansion of CAG repeats in SCA17 locus, suggesting a molecular basis for lack of anticipation in SCA17 families with interrupted CAG expansion.

Myotonic dystrophy type 2 (DM2) is caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the ZNF9 gene on chromosome 3q21. All studied DM2 mutations have been reported in Caucasians and share an identical haplotype, suggesting a common founder. We identified a Japanese patient with DM2 and showed that the affected haplotype is distinct from the previously identified DM2 haplotype shared among Caucasians. These data strongly suggest that DM2 expansion mutations originate from separate founders in Europe and Japan and are more widely distributed than previously recognized.

Spinocerebellar ataxia type 10 (SCA10) is a dominantly inherited neurodegenerative disease caused by expansion of the ATTCT pentanucleotide repeat in intron 9 of a novel gene, ATXN10, on chromosome 22q13.3. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. The length of the expanded ATTCT repeats is highly unstable on paternal transmission and shows a variable degree of somatic and germline instabilty, revealing complex SCA10 genetic mechanisms. How this untranslated ATTCT expansion leads to neurodegeneration has been still controversial. Growing number of evidence indicates a gain-of-function RNA mechanism, similar to the myotonic dystrophies caused by non-coding CTG or CCTG repeat expansions.