Tsunao Saitoh

The precursor protein of non-Aβ component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system

Non-A beta component of Alzheimers disease amyloid (NAC) is the second component in the amyloid from brain tissue of patients affected with Alzheimers disease. Its precursor protein (NACP) was shown to be a brain-specific protein. In rat brain, NACP was more abundant in the neocortex, hippocampus, olfactory bulb, striatum, thalamus, and cerebellum and less abundant in the brain stem. Confocal laser microscopy analysis revealed that anti-NACP immunostaining was colocalized with synaptophysin-immunoreactive presynaptic terminals. Ultrastructural analysis showed that NACP immunoreactivity was associated with synaptic vesicles. NACP sequence showed 95% identity with that of rat synuclein 1, a synaptic/nuclear protein previously identified in rat brain, and good homology with Torpedo synuclein from the electric organ synapse and bovine phosphoneuroprotein 14 (PNP-14), a brain-specific protein present in synapses. Therefore, NACP is a synaptic protein, suggesting that synaptic aberration observed in senile plaques might be involved in amyloidogenesis in Alzheimers disease.

Secreted form of amyloid β protein precursor is involved in the growth regulation of fibroblasts

Fibroblasts that harbor an antisense construct of amyloid beta protein precursor (ABPP) cDNA, A-1, produced less ABPP mRNA and ABPP and grew poorly. Normal growth was restored when either parent cell conditioned medium (CM) or purified ABPP was provided. The capacity of the CM to restore cell growth was abolished by passage through an anti-ABPP immunoaffinity column; the activity was in the bound fraction. A Mr 90,000 protein recognized by the anti-ABPP antibody was diminished in the CM of A-1. CM from ABPP cDNA-transfected cells expressing high levels of ABPP was more potent than that from non-transfected parent cells in restoring A-1 growth. These results indicate that ABPP is released from cells into the medium and has an autocrine function in growth regulation.

The regulation of amyloid β protein precursor secretion and its modulatory role in cell adhesion

The regulation and function of two forms of the amyloid beta protein precursor (ABPP) that are released into the growth-conditioned medium of the PC12 nerve cell line were examined. Nerve growth factor increases the release of the form of ABPP without the protease-inhibitor domain relative to the protein containing the protease inhibitor and increases the overall rate of ABPP secretion 2-fold. In contrast, fibroblast growth factor increases the rate of ABPP secretion approximately 7-fold. Both forms of the secreted ABPP molecule are, in turn, able to stimulate adhesion of PC12 cells to substrata to which they are adsorbed about 10-fold more efficiently on a molar basis than Iaminin.

Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), and apolipoprotein E receptor, with late-onset Alzheimer's disease

The presence of the APOE ϵ4 allele encoding apolipoprotein E4 (apoE4) is the major genetic risk factor for late-onset Alzheimers disease (AD). However, the molecular and cellular mechanisms by which APOE ϵ4 renders AD risk are unclear. In this report, we present genetic evidence that an apoE receptor, LRP, may be associated with the expression of late-onset AD. Using a biallelic genetic marker in exon 3 LRP, late-onset AD cases markedly differed from the control subjects in the distribution of LRP genotypes, and this difference was highly accentuated among AD cases with positive family history of senile dementia. Furthermore, the numbers of neuritic plaques were significantly altered as a consequence of different LRP genotypes in postmortem AD cases. Taken together, our results implicate the pathophysiology of LRP in the expression of late-onset AD.

Decreased levels of protein kinase C in Alzheimer brain

Protein kinase C (PK-C) levels were determined using [3H]phorbol-12,13-dibutyrate (PDB) binding and the in vitro phosphorylation of histone H I (III-S), in autopsied human frontal cortex of age- and postmortem time-matched normal and Alzheimer patients. PK-C levels in Alzheimer particulate fractions determined by both methods were about 50% of those in controls. PK-C levels in Alzheimer cytosol fractions were not significantly different from those in controls. In a parallel study, we measured the phosphorylation of a Mr 86,000 protein (P86), the major protein kinase C substrate in the cytosol fraction prepared from Alzheimer frontal cortex, and found it to be reduced to 43% of that in control brains. This reduction in P86 protein phosphorylation compared to controls was not detected in brain samples prepared from demented patients without Alzheimers disease. We considered 3 extraneous factors (postmortem delay, age and sex) which may have affected the extent of P86 phosphorylation and concluded that the reduced P86 phosphorylation in the Alzheimer samples is not due to any of them. Reduced PK-C levels and Mr 86,000 protein phosphorylation may reflect a biochemical deficit related specifically to the pathogenesis of Alzheimers disease.

Episodic memory changes are associated with the APOE- epsilon 4 allele in nondemented older adults

Objective: To compare the memory performances of nondemented older adults with and without the epsilon 4 allele of the apolipoprotein E (APOE- epsilon 4). Background: Few studies have examined the cognitive status of subjects at high risk for the development of dementia of the Alzheimer type (DAT). A newly reported risk factor for DAT allows for an examination of the cognitive performances of nondemented subjects who are at risk by virtue of being either heterozygous or homozygous for the APOE- epsilon 4 allele. Methods: The California Verbal Learning Test (CVLT) was administered to 52 nondemented older adults. Subjects were divided into two groups on the basis of the presence (n equals 17) or absence (n equals 35) of one or two APOE- epsilon 4 alleles. Results: APOE- epsilon 4 and non- epsilon 4 groups did not significantly differ in demographic, mental status, and functional characteristics. APOE- epsilon 4 subjects demonstrated significantly poorer mean performances than non- epsilon 4 subjects on nine CVLT variables. Seven group differences remained significant, and three approached significance (0.05 less than p less than 0.10), after the effects of age and gender were taken into account. Six of the 14 APOE- epsilon 4 subjects who completed annual follow-up evaluations developed either DAT or questionable DAT, whereas none of the 26 non- epsilon 4 subjects who received follow-up demonstrated any cognitive decline. Conclusions: Results suggest that episodic memory changes in older adults are associated with APOE- epsilon 4 allele; sensitive cognitive markers such as those of the CVLT may precede the subsequent development of DAT. NEUROLOGY 1995;45: 2203-2206

Patterns of aberrant sprouting in alzheimer's disease

Alzheimers disease (AD) is characterized by extensive synaptic and neuronal loss and by plaque formation in the cortex, but the mechanisms responsible for synaptic plasticity in the neocortex are still not completely understood. To analyze the sprouting response in AD cortex, we compared the patterns of GAP-43 with synaptophysin immunoreactivity. In AD, GAP-43 immunohistochemistry revealed extensive sprouting in the hippocampal molecular layer, stratum polymorphous, CA1 region, and prosubiculum. These regions presented abundant anti-GAP-43-immunoreactive coiled fibers and dystrophic neurites in association with plaques. Some of these sprouting structures were colocalized with anti-synapto-physin- and anti-neurofilament-positive neurites. The AD neocortex was characterized by an overall decrease in GAP-43 immunoreactivity accompanied by sprouting neurites in the areas of synaptic pathology. We conclude that GAP-43 might be involved in the mechanisms of synaptic plasticity in the AD cortex, as well as in the process of aberrant sprouting in the neuritic plaques.

The apolipoprotein E epsilon 4 allele is associated with increased neuritic plaques and cerebral amyloid angiopathy in Alzheimer's disease and Lewy body variant

Objective: To determine the relationship between apolipoprotein E (apoE) genotype and neuropathologic lesions in Alzheimers disease (AD) and Lewy body variant (LBV). Design: Retrospective genetic-neuropathologic study of AD and LBV cases. The main neuropathologic outcome measures were modeled as a function of apoE genotype, neuropathologic diagnosis, and gender. Age at death and duration of symptom effects were controlled for by ANCOVA. Patients: One hundred twenty-seven cases with neuropathologically diagnosed AD (n = 84) or LBV (n = 43). Main outcome measures: Quantitative scores of neuritic plaques (NPs), neurofibrillary tangles (NFTs), cerebral amyloid angiopathy (CAA) severity, and CAA prevalence were averaged across four brain regions: midfrontal, inferior parietal, superior temporal, and hippocampal. Results: The apoE epsilon 4 allele was associated with increased NPs within both AD and LBV. The epsilon 4 allele was associated with an increased frequency of CAA in the AD and LBV groups combined and in LBV alone. While CAA severity and NFTs were increased in the epsilon 4/4 homozygous cases when AD and LBV were combined, there were no significant effects within AD or LBV alone. Conclusions: The apoE epsilon 4 allele is strongly associated with increased NPs, but not neocortical NFTs, in both AD and LBV. NEUROLOGY 1996;47: 190-196

Differential involvement of protein kinase C isozymes in Alzheimer's disease

Decreased levels of protein kinase C (PKC) and a reduction in the in vitro phosphorylation of a Mr 86,000 protein (P86), the major PKC substrate, are biochemical characteristics of brain tissue from patients with Alzheimers disease (AD) (Cole et al., 1988). In the current study, we utilized antibodies against individual isozymes of PKC to assess the degree of involvement of different PKC isoforms in AD. The concentration of PKC(beta II) was lower in particulate fractions prepared from AD hippocampal and cortical tissue than in controls and higher in AD cytosol fractions from the cortex than in controls. Immunohistochemical studies in AD neocortex revealed reduced numbers of anti-PKC(beta II)-immunopositive neurons and diminished staining intensity. In contrast, AD hippocampal neurons in CA3-CA4 were more intensely stained with anti-PKC(beta II) antiserum than were controls. The concentration of PKC(beta I) was lower in particulate fractions prepared from AD hippocampus than in controls and was higher in soluble fractions prepared from AD cortex than in controls. The concentration of PKC(alpha) was lower in AD particulate fractions than in controls in the hippocampus. Immunohistochemistry with PKC(alpha) antiserum revealed moderately intense neuron staining and an intense staining of glial cells in AD neocortex. The concentrations and histochemical distributions of PKC(gamma) were not altered in the disease. PKC immunoreactivity was also found in neuritic plaques. The staining patterns of neuritic plaques with different isoform antibodies varied considerably. Anti-PKC(alpha) faintly stained entire plaques and surrounding glial cells; anti-PKC(beta I) stained dystrophic plaque neurites; and anti-PKC(beta II) stained the amyloid-containing portions of plaques.

Expression Pattern of Synucleins (Non‐Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α‐Synuclein) During Murine Brain Development

Abstract: The non‐Aβ component of Alzheimers disease amyloid precursor protein (NACP) is predominantly a neuron‐specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimers disease amyloid and a mutation in the NACP gene is associated with familial Parkinsons disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.