Fumitaka Oyama

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and γ-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that β subunits of voltage-gated sodium channels are sequentially processed by BACE1 and γ-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Nav1.1 α subunits. Sodium channels are modulated by β1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Nav1.1 α subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of β1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b−/− versus Scn1b+/+ mice. Scn1b−/− CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a+/− model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b−/− mice seize spontaneously, the seizure susceptibility of Scn1b+/− mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

Increased expression of p62 in expanded polyglutamine-expressing cells and its association with polyglutamine inclusions

Huntingtons disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60‐kDa protein was increased in Neuro2a cells expressing the N‐terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin‐associated domain‐containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine‐expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine‐expressing cells. We also found that p62 formed aggresome‐like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock‐down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine‐induced stress rather than as a cross‐linker between ubiquitylated proteins.

τ Is Widely Expressed in Rat Tissues

Abstract: The microtubule‐associated protein τ, a major component of paired helical filaments in Alzheimers disease, had been thought to be a neuron‐specific protein. We investigated various rat tissues using both reverse transcriptase‐coupled polymerase chain reaction and immunoblotting. τ was found to be widely expressed in many tissues besides the nervous system: at relatively high levels in the heart, skeletal muscle, lung, kidney, and testis and at low levels in the adrenal gland, stomach, and liver. In terms of the τ isoform expression, tissues fall into three classes: those expressing predominantly small τ, those expressing predominantly big τ, and those expressing both at comparable levels. The phosphorylation state of τ varied among the tissues, as shown by differences in the extents of changes in the reactivities with Tau 1 and electrophoretic mobilities after dephosphorylation. It is notable that τ in many nonneural tissues was highly phosphorylated at Ser396 (according to the numbering of the 441‐residue human τ isoform). Thus, τ is widely expressed in rat tissues.

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.

Association of transferrin C2 allele with late-onset Alzheimer’s disease

Abstract Transferrin (Tf), an iron-transporting protein, has many variants, but C1 and C2 variants account for the majority of the population in all races. Since Tf is reported to be immunocytochemically detectable in senile plaques in Alzheimer’s disease (AD), we have examined the Tf allele frequency among AD patients. The C2 allele frequency in late-onset AD patients is significantly higher than that in age-matched controls. Unexpectedly, the C2 allele frequency in AD patients homozygous for the ApoE ɛ4 allele is markedly increased, i.e., it is twice as high as that in the remaining AD patients carrying zero or one copy of the ɛ4 allele.

Mutant Presenilin 2 Transgenic Mouse: Effect on an Age-Dependent Increase of Amyloid β-Protein 42 in the Brain†

Abstract: The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimers disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild‐type or mutant PS2 under transcriptional control of the chicken β‐actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five‐ to 15‐fold higher than that of endogenous mouse PS2 mRNA. The amyloid β‐protein (Aβ) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild‐type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Aβ42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild‐type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N‐terminal and C‐terminal fragments between wild‐type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age‐dependent increase of Aβ42 content in the brain.

Functional reciprocity between Na+ channel Nav1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

Voltage-gated Na+ channel (VGSC) β1 subunits regulate cell–cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na+ current (INa) mediated by Nav1.6. In addition, β1 is required for high-frequency action potential firing. Transient INa is unchanged in Scn1b (β1) null CGNs; however, the resurgent INa, thought to underlie high-frequency firing in Nav1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Nav1.6 is reduced in Scn1b null cerebellar neurons. In place of Nav1.6 at the AIS, we observed an increase in Nav1.1, whereas Nav1.2 was unchanged. This indicates that β1 is required for normal localization of Nav1.6 at the AIS during the postnatal developmental switch to Nav1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Nav1.6 such that β1-mediated neurite outgrowth requires Nav1.6-mediated INa, and Nav1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

Down's Syndrome: Up-Regulation of β-Amyloid Protein Precursor and τ mRNAs and Their Defective Coordination

Abstract: Almost all patients >40 years of age with Downs syndrome (DS) develop the pathology characteristic of Alzheimers disease: abundant β‐amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of β‐amyloid protein precursor (APR) and τ in DS and age‐matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up‐regulation was not observed in two other neuronal proteins. A correlation between total APP and τ mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP‐695 mRNA and three‐repeat τ mRNA in DS, the proportionality between APP‐751 mRNA and four‐repeat τ mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up‐regulation of τ mRNA as well as APP mRNA and disruption of the coordinate expression between APP‐751 and four‐repeat τ.

Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor

In Huntingtons disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF‐Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF‐Y binding to the promoter region of HSP70, one of the NF‐Y targets. RT–PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF‐Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF‐Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF‐Y could be an important target of mutant Huntingtin.