Biomedical subjects

Shinobu C Fujita

Publications and source records attributed to Shinobu C Fujita.

12 recordsLinked to original sources

PubMed2005

Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord.

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Accessory Nerve↗

PubMed2004

Age-dependent enhancement of hippocampal long-term potentiation in knock-in mice expressing human apolipoprotein E4 instead of mouse apolipoprotein E.

Human apolipoprotein E (apoE) comprises three isoforms, apoE2, apoE3 and apoE4, and apoE4 has been reported as a risk factor of Alzheimer's disease (AD). One of the clinical symptoms of AD is disorder of memory that has been suggested to be related with synaptic plasticity such as long-term potentiation (LTP). Here, we show the enhancement of hippocampal LTP at younger age in knock-in mice lacking mouse apoE, but instead expressing human apoE4. The enhancement of LTP in apoE4 knock-in mice is age-dependent, and it disappears in adult apoE4 knock-in mice. In apoE3 knock-in mice LTP is unaltered, thus human apoE4, but not apoE3, specifically modulates synaptic plasticity at younger age. Since basal synaptic transmission and distribution of glutamate receptors, as well as presynaptic functions, are intact in apoE4 knock-in mice, postsynaptic functional modification of LTP through lipid homeostasis is suggested. ApoE4 knock-in mice would be a useful animal model of human apoE4 carriers, and our finding that LTP is enhanced in younger apoE4 knock-in mice is in accord with the previous report showing higher intelligence in young human apoE4 carriers.

2-Amino-5-phosphonovalerate↗

PubMed2004

Phosphorylated alpha-synuclein in normal mouse brain.

alpha-Synuclein phosphorylated at Ser129 is the main component of Lewy bodies of Parkinson's and closely related diseases. We studied, by quantitative immunoblotting, changes in the phosphorylation level of alpha-synuclein in the mouse brains subjected to cold water stress. Relative basal level of alpha-synuclein phosphorylation at Ser129 was 40% higher in the striatum compared with the hippocampus. The phosphorylation level decreased to 57% in the striatum 20 min after 5 min of cold water stress, and also in the hippocampus and cortex to lesser degrees. Recovery to basal levels took place over several hours. The stress-induced temporary dephosphorylation was of smaller magnitude in the striatum of aged (18 months) mice. These results show that alpha-synuclein phosphorylation level at Ser129 in vivo responds to physiological stimuli. Relative prominence and age sensitivity of this phenomenon in the striatum may be relevant to the pathogenesis of Parkinson's disease.

Aging↗

PubMed2004

Accelerated Abeta aggregation in the presence of GM1-ganglioside-accumulated synaptosomes of aged apoE4-knock-in mouse brain.

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4- than the apoE3-knock-in mouse brain. Furthermore, we show that Abeta aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.

Aging↗

PubMed2004

Augmented delayed infarct expansion and reactive astrocytosis after permanent focal ischemia in apolipoprotein E4 knock-in mice.

Using homozygous human apolipoprotein E2 (apoE2) (2/2)-, apoE3 (3/3)-, or apoE4 (4/4)-knock-in (KI) mice, we aimed to examine whether an apoE isoform-specific exacerbation of delayed infarct expansion occurs after permanent middle cerebral artery occlusion (pMCAO). Compared with 2/2- or 3/3-KI mice, 4/4-KI mice exhibited significantly larger infarct volumes and worse neurologic deficits after pMCAO, with no significant differences between the latter two groups. Infarct volume in 4/4-KI mice was significantly increased from 1 to 5 days after pMCAO, whereas that in 2/2- or 3/3-KI mice was not significantly altered. DNA fragmentation in the peri-infarct area as detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatenick end-labeling was increased to a similar degree in all of the KI mice by 5 days after pMCAO, with no significant differences among the mouse groups. At every time-point examined, human apoE was most markedly expressed in the peri-infarct area, with similar immunoreactivity among the three lines of KI mice. The glial fibrillary acidic protein immunoreactive burden in the peri-infarct area was progressively increased through 7 days in 4/4-KI mice, but not in 2/2- or 3/3-KI mice. Taken together, these data show that the apoE4 isoform acts to aggravate delayed infarct expansion and peri-infarct reactive astrocytosis during the subacute phase of pMCAO in genetically engineered apoE-KI mice.

Animals↗

PubMed2003

Phosphorylation state of tau in the hippocampus of apolipoprotein E4 and E3 knock-in mice.

The apolipoprotein E (apoE) epsilon 4 allele is associated with an increased risk of sporadic as well as late-onset familial Alzheimer's disease (AD). To accurately determine the isoform-specific effects of human apoE on AD-like phosphorylation of tau, hippocampi from human apoE knock-in (KI) mice were studied by quantitative immunoblotting. There was no significant difference in phosphorylation levels of tau at nine of the 13 epitopes, for six of eight tau kinases, or in protein levels of three tau phosphatases, between apoE3-KI and apoE4-KI mouse hippocampi. However, in apoE4-KI mice, phosphorylation of tau at Ser235 was increased to approximately 150%, that at Ser413 to approximately 140%, while that at Ser202/Thr205 and Thr205 were decreased to approximately 70%, and the protein level of tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK3beta) was increased to approximately 120%, that of extracellular signal-regulated kinase 2 (ERK2) was increased to approximately 130%, compared with apoE3-KI mice.

Animals↗

PubMed2003

Stress-induced hyperphosphorylation of tau in the mouse brain.

We previously showed that starvation causes reversible hyperphosphorylation of tau in the mouse brain. To explore possible involvement of stress in tau hyperphosphorylation quantitative analysis of phosphorylated tau in four brain regions of mice subjected to cold water stress (CWS) was made by immunoblot analyses using phosphorylation-dependent antibodies directed to eight sites on tau known to be hyperphosphorylated in the brain of Alzheimer's disease (AD) patients. Ser199, Ser202/Thr205, Thr231/Ser235 were hyperphosphorylated 20 and 40 min after CWS. The response was pronounced in the hippocampus and cerebral hemisphere, but weak in the cerebellum in parallel with the regional vulnerability in AD. Among the regulatory phosphorylation of protein kinases studied, a transient phosphorylation of tau protein kinase I/glycogen synthase kinase 3beta at Ser9 was most conspicuous.

Animals↗

PubMed2003

MuSC is involved in regulating axonal fasciculation of mouse primary vestibular afferents.

Regulation of axonal fasciculation plays an important role in the precise patterning of neural circuits. Selective fasciculation contributes to the sorting of different types of axons and prevents the misrouting of axons. However, axons must defasciculate once they reach the target area. To study the regulation of fasciculation, we focused on the primary vestibulo-cerebellar afferents (PVAs), which show a dramatic change from fasciculated axon bundles to defasciculated individual axons at their target region, the cerebellar primordium. To understand how fasciculation and defasciculation are regulated in this system, we investigated the roles of murine SC1-related protein (MuSC), a molecule belonging to the immunoglobulin superfamily. We show: (i) by comparing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) labelling and anti-MuSC immunohistochemistry, that downregulation of MuSC in PVAs during development is concomitant with the defasciculation of PVA axons; (ii) in a binding assay with cells expressing MuSC, that MuSC has cell-adhesive activity via a homophilic binding mechanism, and this activity is increased by multimerization; and (iii) that MuSC also displays neurite outgrowth-promoting activity in vestibular ganglion cultures. These findings suggest that MuSC is involved in axonal fasciculation and its downregulation may help to initiate the defasciculation of PVAs.

Aging↗

PubMed2003

Increased vulnerability to focal ischemic brain injury in human apolipoprotein E4 knock-in mice.

Accumulating evidence suggests that among the 3 human apolipoprotein E (apoE) isoforms encoded by the human APOE gene, the e4 allele may act to exacerbate brain damage in humans and animals. This study aimed to compare the isoform-specific vulnerability conferred by human apoE to ischemic brain damage, using mice expressing human apoE isoforms (apoE2, apoE3, or apoE4) in place of mouse apoE, produced by the gene-targeting technique in embryonic stem cells (knock-in, KI). Homozygous human apoE2 (2/2), apoE3 (3/3), or apoE4 (4/4) KI mice were subjected to permanent focal cerebral ischemia by a modified intraluminal suture method. Twenty-four h thereafter, brain damage, (as estimated by infarct volume and neurologic deficit) was significantly worse in 4/4 KI mice versus 2/2 or 3/3 KI mice (p < 0.001 for each comparison), with no significant differences between 2/2 and 3/3 KI mice. Immunohistochemistry for human apoE expression revealed similar apoE distribution with no significant difference in the immunostaining intensity among the 3 lines of KI mice. Notably. increased expression of human apoE was detected in neurons and astrocytes in the peri-infarct area, and a punctate expression pattern was evident in the border between the infarct and peri-infarct areas in all KI mice subjected to ischemia. Taken together, our results show that apoE affects the outcome of acute brain damage in an isoform-specific fashion (apoE4 > apoE3 = apoE2) in genetically engineered mice.

Animals↗

PubMed2002

Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice.

We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K. (2000) J. Neurochem. 74, 1008-1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner. However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined. In this study, we demonstrate that the cholesterol release is mediated by endogenously synthesized and secreted apoE isoforms and clarify the mechanism underlying this apoE isoform-dependent cholesterol release using cultured astrocytes prepared from human apoE3 and apoE4 knock-in mice. Cholesterol and phospholipids were released into the culture media, resulting in the generation of two types of high density lipoprotein (HDL)-like particles; one was associated with apoE and the other with apoJ. The amount of cholesterol released into the culture media from the apoE3-expressing astrocytes was approximately 2.5-fold greater than that from apoE4-expressing astrocytes. In contrast, the amount of apoE3 released in association with the HDL-like particles was similar to that of apoE4, and the sizes of the HDL-like particles released from apoE3- and apoE4-expressing astrocytes were similar. The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3- and apoE4-expressing astrocytes were 250 +/- 6.0 and 119 +/- 5.1, respectively. These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes. These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.

Animals↗

PubMed2002

Cholesterol is increased in the exofacial leaflet of synaptic plasma membranes of human apolipoprotein E4 knock-in mice.

Inheritance of the apolipoprotein (apoE) epsilon4 allele is a risk factor for developing Alzheimer's disease (AD). The purpose of the present study was to determine effects of apoE-isoforms on the transbilayer distribution of cholesterol in synaptic plasma membranes (SPM) using mice expressing human apoE3 and apoE4. Total SPM cholesterol levels did not differ among the wild-type and apoE3 and apoE4 knock-in mice. However, a striking difference was observed in the transbilayer distribution of SPM cholesterol. ApoE4 knock-in mice showed an approximately 2-fold increase in exofacial leaflet cholesterol compared with apoE3 knock-in mice and wild-type mice. The results of this study suggest that pathogenic effects of apoE4 on AD development could be closely linked to alteration of cholesterol distribution in SPM.

Alzheimer Disease↗

PubMed2002

Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3beta.

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer-like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI)/GSK3beta with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2/GSK3beta2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI/GSK3beta isoforms within young neurons. TPKI2/GSK3beta2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2/GSK3beta2 occurs predominantly in the neuronal soma, while TPKI1/GSK3beta1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk-2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI/GSK3beta.

Alternative Splicing↗