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To confirm the intracellular accumulation of amyloid beta-protein (Abeta), we carefully performed immunohistochemistry using brains of cynomolgus monkeys of various ages. Cortical neurones and their large neurites were immunostained with antibodies against Abeta in young monkey brains. In aged monkey brains, intracellular Abeta localized within cortical neurones; no clear association was found between the presence of intracellular Abeta and senile plaques (SPs). Interestingly, we did not observe Abeta-immunoreactive cortical neurones in brains fixed with neutral buffered formalin. Western blot analyses of microsomal and nerve ending fractions derived from the brains of young to aged monkeys revealed that intracellular Abeta generation changed with age. In the microsomal fraction, the amount of Abeta42 significantly increased in brains from older monkeys (>30 years of age), and the amount of Abeta43 significantly decreased with age in the microsomal fraction. The amount of Abeta40 remained the same regardless of age. Biochemical analyses also showed that intracellular levels of each of these Abeta molecules significantly increased with age in nerve ending fractions. As we previously observed that a similar accumulation of presenilin1, beta-amyloid precursor protein (APP) and APP C-terminal fragment cleaved by beta-secretase in the nerve ending fractions obtained from brains with SPs, the accumulation of intracellular Abeta in this fraction may be closely related to formation of spontaneous SPs with age. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending will contribute to a better understanding of the pathogenesis of age-related neurodegenerative disorders such as sporadic Alzheimer's disease.

We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains and also examined age-related biochemical changes of Alzheimer's disease (AD)-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (>20 years old), we found SPs but no neurofibrillary tangles (NFTs). Antibodies against beta-amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs; however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4-years-old), the expression of full-length APP751 protein was increased in brains from older monkeys (>20 years old). Biochemical analyses also showed that levels of various AD-associated proteins increased significantly with age in nerve ending fractions. Both SP-associated (APP) and NFT-associated proteins (tau, activated glycogen synthase kinase 3beta, cyclin dependent kinase 5, p35, and p25) accumulated in the nerve ending fraction with increasing age; however, we found no NFTs or paired helical filaments of tau in aged cynomolgus monkey brains. This age-related accumulation of these proteins in the nerve ending fraction was similar to that observed in our laboratory previously for presenilin-1 (PS-1). The accumulation of these SP-associated proteins in this fraction may be a causal event in the spontaneous formation of SPs; thus, SPs may be formed initially in nerve endings. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending and in axonal transport will contribute to a better understanding of the pathogenesis of neurodegenerative disorders such as AD.

Infection with JHMV results in the transcriptional activation of two host cell genes encoding proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta. Analysis of irradiated mice showed that IL-1 beta mRNA accumulation in the central nervous system was predominantly derived from the mononuclear infiltrate. By contrast, accumulation of TNF-alpha mRNA was unaffected by immunosuppression, suggesting that resident cells were the source of this cytokine. Infected mice were treated with anti-TNF antibody to determine if TNF-alpha contributed to either the encephalomyelitis or demyelination associated with JHMV infection. Surprisingly, neither the cellular infiltrate nor demyelination were affected. In vitro analysis showed that IL-1 beta but not TNF was secreted from JHMV infected macrophages. The absence of TNF secretion is due to a block in translation of the TNF mRNA which accumulates during infection.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.

Spleen cells cultured from Balb/c mice immunized with the JHM strain of mouse hepatitis virus (JHMV) have CD8+ cytotoxic T cells (CTL) specific for both the S and N proteins, but not the M or HE proteins. T cell lines were established from the brains of Balb/c mice infected with JHMV. The majority of the lines (20 of 22) were specific for JHMV. Analysis of the viral structural proteins which served as target structures indicate that most (15 of 20) were specific for the N protein. One line was specific for the S protein and four lines were specific for JHMV but the protein recognized could not be determined. These data suggest that early during infection there is a preferential recruitment of N protein specific CTL into the CNS of infected mice.