Kinji Ohno

Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence

A mutant mitochondrial DNA (mtDNA) with a 4,977-bp deletion was detected in the parkinsonian brain by using the polymerase chain reaction. Although the deleted mtDNA was detectable even in the brain of aged controls, the proportion of deleted mtDNA to normal mtDNA in the striatum was higher in the parkinsonian patients than in the controls. In both the parkinsonian patients and the aged controls, the proportion was higher in the striatum than in the cerebral cortex. These results indicate that age-related accumulation of deleted mtDNA is accelerated in the parkinsonian striatum and suggest that the deletion contributes to pathophysiological processes underlying Parkinsons disease.

Mutation of the acetylcholine receptor α subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Congenital Myasthenic Syndrome Caused by Decreased Agonist Binding Affinity Due to a Mutation in the Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Rapsyn Mutations in Humans Cause Endplate Acetylcholine-Receptor Deficiency and Myasthenic Syndrome

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.

Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis

Deleted mitochondrial DNA (mtDNA) was accumulated in the parkinsonian striatum, but the same deleted mtDNA was also detectable in the control striatum when cycles of polymerase chain reaction were increased. To discriminate between these pathological and physiological conditions, we quantitatively analyzed the proportion of deleted mtDNA to normal mtDNA by measuring the incorporation of alpha-[32P]deoxycytosine triphosphate into mtDNA fragments by using a laser image analyzer. To estimate the molar ratio of the deleted mtDNA to normal mtDNA, the radioactivity was normalized by each fragment size. By plotting logarithms of normalized radioactivities against PCR amplification cycles, straight lines were obtained with different slopes. By extrapolation of the line to the zero amplification, the proportion of mutant mtDNA to normal mtDNA in the original sample from the parkinsonian striatum was estimated to be ca. 5%, which was at least ten times higher than the proportion of ca. 0.3% in the control striatum. These results indicate that phenotype of the mutant mtDNA as Parkinsons disease is expressed when the proportion of deleted mtDNA to normal mtDNA exceeds a threshold of ten times higher value than in the normal subject.

Multiple mitochondrial DNA deletions exist in cardiomyocytes of patients with hypertrophic or dilated cardiomyopathy

Genetic impairment was revealed in idiopathic cardiomyopathy and the responsible DNA locus was estimated. Mitochondrial DNA were amplified from autopsied cardiac specimens from three patients who died from hypertrophic or dilated cardiomyopathy by using polymerase chain reaction (PCR). By using two novel methods for PCR gene amplification, the pleioplasmic existence of multiple populations of differently deleted mitochondrial DNA in all specimens from the patients was confirmed. Mitochondrial DNA with a 7,436 bp deletion which commonly existed among the specimens was sequenced and the direct repeat at each edge of deletion was identified as (CATCAACAACCG) which was located in ATPase 6 gene and in the D-loop region. From our results mitochondrial DNA mutations could also be an important contributory factor to cardiomyopathy.

Human branch point consensus sequence is yUnAy

Yeast carries a strictly conserved branch point sequence (BPS) of UACUAAC, 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 yUnAy, 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 21–34 nucleotides upstream of the 3′ end of an intron in 83% clones. We also found that the polypyrimidine tract spans 4–24 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.

Sleuthing molecular targets for neurological diseases at the neuromuscular junction

The analysis of congenital myasthenic syndromes (CMSs) has disclosed a diverse array of molecular targets at the motor endplate and has delineated their contribution to synaptic function. Clinical, electrophysiological and morphological studies have paved the way for detecting CMS-related mutations in proteins such as choline acetyltransferase, acetylcholinesterase, the acetylcholine receptor and rapsyn, and studies of the mutant proteins have allowed us to correlate the effects of the mutations with predicted alterations in protein structure. Here, we review the symptomatology of CMSs, consider the factors that impair neuromuscular transmission, survey the mutations that have been uncovered in the different synaptic proteins, and consider the functional implications of the identified mutations.

Congenital myasthenic syndromes: progress over the past decade.

Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic basal lamina, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A basal lamina CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow‐ and fast‐channel syndromes, respectively. Most low‐expressor mutations reside in the AChR ϵ subunit and are partially compensated by residual expression of the fetal type γ subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane. Muscle Nerve 27: 4–25, 2003

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.