It has been shown that human monocytes express rnonocyte chemoattractant protein-1 (MCP-1), an inflammatry factor, in response to non-fibrillar P-amyloid protein. Reactive niicroglia and inflammatory factors were reported to be present in P-amyloid deposits (senile plaques) in Alzheimer's disease, suggesting the presence of MCP-1 in senile plaques. To address this issue, we examined MCP-1-immunoreactivity in senile plaques using a mouse monoclonal anti-MCP-1 antibody. Monocyte chemoattractant protein-1 was found immunohistochemically in mature senile plaques and reactive microglia but not in immature senile plaques of brain tissues from five patients with Alzheimer's disease. These findings suggest that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques.

Abstract:To examine the physiological roles of the ␦ subunit of Ca 2ϩ /calmodulin-dependent protein kinase II (CaM kinase II␦) in brain, we examined the localization of CaM kinase II␦ in the rat brain. A specific antibody to CaM kinase II␦1-␦4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the ␣, ␤, and ␥ subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase II␦1-␦4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products ␦3.1, ␦3.4, and ␦3 that contained the nuclear localization signal. These CaM kinase II␦3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase II␦3 isoforms are involved in nuclear Ca 2ϩ signaling in cerebellar granule cells. Key Words: Calmodulin-Ca 2ϩ /calmodulindependent protein kinase II-Cerebellum-Granule cells-Nucleus-Overexpression. J. Neurochem. 72, 815-825 (1999). Ca2ϩ /calmodulin-dependent protein kinase II (CaM kinase II) is highly concentrated in neuronal tissues and may be related to many neuronal functions (for review, see Colbran et al., 1989;Hanson and Schulman, 1992;Fukunaga et al., 1996). Four subunits, termed ␣, ␤, ␥, and ␦, are encoded by distinct genes in eukaryotes Schulman and Hanson, 1993). The ␥ and ␦ subunits appear to have widespread distribution, whereas the ␣ and ␤ subunits are predominant forms in the brain. All subunits are highly conserved and have Ͼ90% amino acid sequence homology in the Nterminal catalytic domain and in the regulatory domain comprising calmodulin-binding and autoinhibitory sequences. The various isoforms differ mainly at the end of the regulatory domain, an area with a variable number of amino acids (variable domain 1). The important findings were that 11-amino acid insertion in variable domain 1 functioned as a nuclear localization signal (NLS) (Srinivasan et al., 1994). Among the subunits of CaM kinase II found in the brain, only the ␣B subunit (corresponding to the ␣-33 subunit in monkey brain) and the ␥A subunit in rat brain had the NLS (Tobimatsu et al., 1988;Benson et al., 1991;Brocke et al., 1995).Northern blot analysis indicated that a significantly higher amount of the ␦ subunit of CaM kinase II (CaM kinase II␦) was expressed in cerebellum than in cerebrum and brainstem (Tobimatsu and Fujisawa, 1989). In situ hybridization histochemistry revealed intense expression signals for CaM kinase II␦ in the cerebellar granule cell layer, but not in the Purkinje cell layer (Takaishi et al., 1992;Sakagami and Kondo, 1993). These results suggest that CaM kinase II␦ was mainly expressed in ...

The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.

SUMMARY:We report a novel localized amyloidosis associated with lactoferrin. To elucidate the precursor protein of corneal amyloidosis associated with trichiasis, we analyzed amyloid deposits from three patients by histopathology and biochemistry. Amyloid deposits showed immunoreactivity, confirmed by electron microscopy, for only anti-human lactoferrin antibody. Electrophoresis of amyloid fibrils revealed lactoferrin with and without sugar chains; N-terminal sequence analysis revealed full-length lactoferrin and a truncated tripeptide of N-terminal amino acids, Gly-Arg-Arg. Carboxymethylated wild-type lactoferrin formed amyloid fibrils in vitro. Lactoferrin gene analysis in the three patients revealed a Glu561Asp mutation in all of the patients and a compound heterozygote of Ala11Thr and Glu561Asp mutations in one patient. A heterozygotic Glu561Asp mutation appeared in 44.8% of healthy Japanese volunteers, suggesting that the mutation may not be an essential mutation for amyloid formation (p ϭ 0.104). Results thus suggest that lactoferrin is this precursor protein. (Lab Invest 2002, 82:757-765).

Several parts of the cerebral cortices in five brains from patientswith Alzheimer's disease were examined by light and electron microscopes. The results obtained are as follows: The initial change of the cerebral cortex in the brain occurred in the small blood vessel and capillary. The endothelial cell of the blood vessel fell into a degenerated state with swelling of the vascular feet and astroglial cells. The change in a great number of nerve cells and their processes diffusely observed in the cortices were nonspecific andcould be due to primary vascular degeneration. Severe dementia in Alzheimer's disease seemed to be well explained by the histo‐pathological findings of diffusely destroyed nerve cells and their processes. From this, Alzheimer's disease can be speculated to be a disease caused by progressive capillary degeneration.