Shun Shimohama

Mitochondrial Abnormalities in Alzheimer's Disease

Mitochondria from persons with Alzheimers disease (AD) differ from those of age-matched control subjects. Differences in mitochondrial morphology and function are well documented, and are not brain-limited. Some of these differences are present during all stages of AD, and are even seen in individuals who are without AD symptoms and signs but who have an increased risk of developing AD. This chapter considers the status of mitochondria in AD subjects, the potential basis for AD subject mitochondrial perturbations, and the implications of these perturbations. Data from multiple lines of investigation, including epidemiologic, biochemical, molecular, and cytoplasmic hybrid studies, are reviewed. The possibility that mitochondria could potentially constitute a reasonable AD therapeutic target is discussed, as are several potential mitochondrial medicine treatment strategies.

Activation and Involvement of p38 Mitogen-activated Protein Kinase in Glutamate-induced Apoptosis in Rat Cerebellar Granule Cells

In the mammalian central nervous system glutamate is the major excitatory neurotransmitter and plays a crucial role in plasticity and toxicity of certain neural cells. We found that glutamate stimulated activation of p38 and stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase (JNK)), two subgroup members of the mitogen-activated protein kinase superfamily in matured cerebellar granule cells. The p38 activation was largely mediated by N-methyl-d-aspartate receptors. Furthermore, we have revealed a novel signaling pathway, that is, Ca2+-mediated activation of p38 in glutamate-treated granule cells. The glutamate concentration effective for inducing apoptosis correlated with that for inducing p38 activation. SB203580, a specific inhibitor for p38, inhibited glutamate-induced apoptosis. Thus p38 might be involved in glutamate-induced apoptosis in cerebellar granule cells.

α7 Nicotinic Receptor Transduces Signals to Phosphatidylinositol 3-Kinase to Block A β-Amyloid-induced Neurotoxicity

Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimers disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against β-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an α7 nicotinic receptor antagonist (α-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The α7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the α7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.

Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia.

Abstract: Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer‐type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (–)‐[3H]nicotine and [3H]quinuclidinyl benzilate ([3H]QNB), respectively. Compared with the controls, (–)‐[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.

Microglial activation and amyloid-β clearance induced by exogenous heat-shock proteins

Alzheimers disease (AD) is characterized by the accumulation of fibrillar amyloid‐β (Aβ) peptides to form amyloid plaques. Understanding the balance of production and clearance of Aβ peptides is the key to elucidating amyloid plaque homeostasis. Microglia in the brain, associated with senile plaques, are likely to play a major role in maintaining this balance. Here, we show that heat‐shock proteins (HSPs), such as HSP90, HSP70, and HSP32, induce the production of interleukin 6 and tumor necrosis factor α and increase the phagocytosis and clearance of Aβ peptides. This suggests that microglial interaction with Aβ peptides is highly regulated by HSPs. The mechanism of microglial activation by exogenous HSPs involves the nuclear factor KB and p38 mitogen‐activated protein kinase pathways mediated by Toll‐like receptor 4 activation. In AD brains, levels of HSP90 were increased in both the cytosolic and membranous fractions, and HSP90 was colocalized with amyloid plaques. These observations suggest that HSP‐induced microglial activation may serve a neuroprotective role by facilitating Aβ clearance and cytokine production.

Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons

It has been shown that estrogen replacement in menopausal women is effective in slowing down the progression of cognitive impairment in Alzheimers disease. Although recent studies have demonstrated the neuroprotective effects of estrogen, the precise mechanism of neuroprotection has not been elucidated. In the present study, we show that the phosphatidylinositol 3‐kinase (PI3‐K) cascade is involved in the neuroprotective mechanism stimulated by estrogen. Exposure to glutamate reduced the viability of rat primary cortical neurons. Pretreatment with 10 nM 17β‐estradiol significantly attenuated the glutamate‐induced toxicity. This neuroprotective effect of 17β‐estradiol was blocked by co‐administration with LY294002, a selective PI3‐K inhibitor, but not by co‐administration with PD98059, a selective mitogen activated protein kinase kinase inhibitor. Pretreatment with ICI182780, a specific estrogen receptor antagonist, also blocked the neuroprotection. Immunoblotting assay revealed that treatment with 17β‐estradiol induced the phosphorylation of Akt/PKB, an effector immediately downstream of PI3‐K. These results suggest that PI3‐K mediates the neuroprotective effect of 17β‐estradiol against glutamate‐induced neurotoxicity. J. Neurosci. Res. 60:321–327, 2000

Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death.

Oxidative stress is important in the process of dopaminergic neuronal degeneration in Parkinsons disease. Recent studies suggest that estrogens have neuroprotective effects in neurodegenerative disorders, including Alzheimers disease. In the present study, we investigated neuroprotection against oxidative stress afforded by estradiol using primary neuronal culture of the rat ventral mesencephalon. Oxidative stress induced by glutamate, superoxide anions, and hydrogen peroxide caused significant neuronal death. Although simultaneous administration of 17β‐estradiol and glutamate did not show any significant effects, preincubation with 17β‐estradiol provided significant neuroprotection against glutamate‐induced neurotoxicity (ED50 was 50 μM for dopaminergic and 15 μM for nondopaminergic neurons). Neuroprotection occurred even after a brief preincubation with 17β‐estradiol and was not significantly blocked by either an estrogen receptor antagonist or a protein synthesis inhibitor. These findings indicate that the neuroprotection against glutamate neurotoxicity is mediated by neither estrogen receptors nor activation of genome transcription. Other steroids (corticosterone, testosterone, and cholesterol) did not provide significant neuroprotection against glutamate‐induced neurotoxicity. Furthermore, preincubation with 17β‐estradiol provided neuroprotection against neuronal death induced by both superoxide anions and hydrogen peroxide. Dichlorofluorescin diacetate, a marker of oxygen radicals, revealed that preincubation with 17β‐estradiol suppressed intracellular oxygen radicals induced by hydrogen peroxide. The biologically inactive stereoisomer of estradiol, 17α‐estradiol, provided neuroprotection against glutamate‐induced toxicity in dopaminergic neurons, as well as the17β isoform. 17α‐estradiol may be a potential therapeutic agent used to prevent dopaminergic neuronal death induced by oxidative stress in Parkinsons disease. J. Neurosci. Res. 54:707–719, 1998.

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease.

Recently, apoptosis has been implicated in the selective neuronal loss of Alzheimers disease (AD). Apoptosis is regulated by the B cell leukemia-2 gene product (Bcl-2) family (Bcl-2, Bcl-x, Bax, Bak and Bad) and the caspase family (ICH-1 and CPP32), with apoptosis being prevented by Bcl-2 and Bcl-x, and promoted by Bax, Bak, Bad, ICH-1 and CPP32. In the present study, we examined the levels of these proteins in the membranous and cytosolic fractions of temporal cortex in AD and control brain. In the membranous fraction, the levels of Bcl-2 alpha, Bcl-xL, Bcl-x beta, Bak and Bad were increased in AD. In the cytosolic fractions, the level of Bcl-x beta was increased, while Bcl-xL, Bax, Bak, and Bad and ICH-1L were unchanged. CPP32 was not detected in AD or control brain. These findings demonstrate a differential involvement of cell death-regulatory proteins in AD and suggest that Bak, Bad, Bcl-2 and Bcl-x are upregulated in AD brains.

Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.