Investigations on oxidative stress and therapeutical implications in dementia

Alzheimer’s disease (AD) is a progressive dementia affecting a large proportion of the aging population. The histopathological changes in AD include neuronal cell death and formation of amyloid plaques and neurofibrillary tangles (NFTs) NFTs are composed of hyperphosphorylated tau protein, and senile plaques contain aggregates of the β-peptide. There is also evidence that brain tissue in patients with AD is exposed to oxidative stress during the course of the disease. Advanced glycation endproducts (AGEs), which are formed by a non-enzymatic reaction of glucose with long-lived protein deposits, are potentially toxic to the cell, are present in brain plaques in AD, and its extracellular accumulation in AD may be caused by an accelerated oxidation of glycated proteins. The microtubuli-associated protein tau is also subject to intracellular AGE formation. AGEs participate in neuronal death causing direct (chemical) radical production: Glycated proteins produce nearly 50-fold more radicals than non-glycated proteins, and indirect (cellular) radical production: Interaction of AGEs with cells increases oxidative stress. During aging cellular defence mechanisms weaken and the damages to cell constituents accumulate leading to loss of function and finally cell death. The development of drugs for the treatment of AD remains at a very unsatisfying state. However, pharmacological approaches which break the vicious cycles of oxidative stress and neurodegeneration offer new opportunities for the treatment of AD. Theses approaches include AGE-inhibitors, antioxidants, and anti-inflammatory substances, which prevent radical production. AGE inhibitors might be able to stop formation of AGE-modified β-amyloid deposits, antioxidants are likely to scavenge intracellular and extracellular superoxide radicals and hydrogen peroxide before these radicals damage cell constituents or activate microglia, and anti-inflammatory drugs attenuating microglial radical and cytokine production.     

Immunohistochemical study of the expression of cytokines and nitric oxide synthases in brains of patients with dementia with Lewy bodies

Regional expression of cytokines (IL‐1α, TNF‐α), inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) was immunohistochemically investigated in the brains of patients with dementia with Lewy bodies (DLB), compared with those of patients with Alzheimer's disease (AD) and non‐demented elderly persons. It has been reported that inflammatory responses by cytokines and oxygen free radicals such as nitric oxide (NO) are associated with damaged neurons, degenerative neurites or amyloid deposits in AD brains. In the present study, overexpression of IL‐1α, TNF‐α and iNOS was demonstrated in the amygdala, hippocampus, entorhinal and insular cortices of DLB brains, which are pathologically the most vulnerable regions in DLB brains as well as AD brains. In addition, some Lewy body (LB)‐bearing neurons were involved by the processes of IL‐1α‐ and TNF‐α‐positive microglia, and most extracellular LB were associated with the processes of TNF‐α‐ and iNOS‐positive astroglia. Glial involvement was also found around neuritic plaques and extracellular neurofibrillary tangles. In contrast, the expression of nNOS was reduced in the amygdala of DLB brains showing severe Lewy pathology. These findings suggest that cytokines and NO are significantly implicated in neuronal damage and death including LB formation in DLB brains.     

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimer's disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.     

Neuronal and glial coexpression of argininosuccinate synthetase and inducible nitric oxide synthase in Alzheimer disease

The enzyme argininosuccinate synthetase (ASS) is the rate limiting enzyme in the metabolic pathway leading from L-citrulline to L-arginine, the physiological substrate of all isoforms of nitric oxide synthases (NOS). ASS and inducible NOS (iNOS) expression in neurons and glia was investigated by immunohistochemistry in brains of Alzheimer disease (AD) patients and nondemented, age-matched controls. In 3 areas examined (hippocampus, frontal, and entorhinal cortex), a marked increase in neuronal ASS and iNOS expression was observed in AD brains. GFAP-positive astrocytes expressing ASS were not increased in AD brains versus controls, whereas the number of iNOS expressing GFAP-positive astrocytes was significantly higher in AD brains. Density measurements revealed that ASS expression levels were significantly higher in glial cells of AD brains. Colocalization of ASS and iNOS immunoreactivity was detectable in neurons and glia. Occasionally, both ASS-and iNOS expression was detectable in CD 68-positive activated microglia cells in close proximity to senile plaques. These results suggest that neurons and astrocytes express ASS in human brain constitutively, whereas neuronal and glial ASS expression increases parallel to iNOS expression in AD. Because an adequate supply of L-arginine is indispensable for prolonged NO generation, coinduction of ASS enables cells to sustain NO generation during AD by replenishing necessary supply of L-arginine.     

Advanced glycation end products co-localized with astrocytes and microglial cells in Alzheimer’s disease brain

   In the previous study [Takeda et al. (1996) Neurosci Lett 221: 17–21], we reported that the advanced glycation end products (AGEs) in the external space of neuronal perikarya (extraneuroperikaryal AGE deposits) were significantly abundant in the Alzheimer’s brain. In this study, we investigated the spatial relationship of the extraneuroperikaryal AGE (carbocymethyllysine and pentosidine) deposits in astrocytes and microglial cells in the Alzheimer’s disease brain using double immunolabelling for AGEs and astrocyte or microglial cell markers. Most of the extraneuroperikaryal AGE deposits were co-localized with glial fibrillary acidic protein-positive astrocytes. AGE deposit-bearing astrocytes also contained Gomori-positive granules. Furthermore, some of the extraneuroperikaryal AGE deposits were co-localized with microglial cells. These extraneuroperikaryal AGEs may activate astrocyte and microglia, and play a role in pathogenesis of Alzheimer’s disease. Received: 8 September 1997 / Revised: 19 November 1997 / Accepted: 12 February 1998     

Distribution of amyloid beta protein precursor in the Alzheimer's disease brain

In order to clarify the distribution and pathological changes of the amyloid beta protein precursor (betaAPP), 10 Alzheimer's disease (AD) brains and seven normal control brains were examined by immunocytochemistry and in situ hybridization histochemistry. All betaAPP isoforms were distributed evenly in neuronal cell bodies and their axons and dendrites. The betaAPP-positive neuronal processes showed mesh-like networks. In AD brains, betaAPP-positive neurons and mesh-like networks were generally decreased in spite of some intensely labeled neurons. All betaAPP isoforms accumulated in neuronal processes, dystrophic neurites and senile plaques. In situ hybridization histochemistry confirmed that all isoforms of betaAPP were expressed in neurons in control brains. In AD brains, the betaAPP mRNA signal was generally decreased besides some intense signal neurons corresponding to immunostaining findings. Few astrocytes expressed betaAPP. Thus, uniform expression and distribution of betaAPP were disturbed in AD brains showing uneven decreases or increases of neuronal betaAPP expression in individual neurons and betaAPP accumulation in neurons, neuronal processes and abnormal structures including dystrophic neurites, senile plaques and neurofibrillary tangles.     

Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains

beta-Amyloid(1-42) (A beta 42), a major component of amyloid plaques, accumulates within pyramidal neurons in the brains of individuals with Alzheimer's disease (AD) and Down syndrome. In brain areas exhibiting AD pathology, A beta 42-immunopositive material is observed in astrocytes. In the present study, single- and double-label immunohistochemistry were used to reveal the origin and fate of this material in astrocytes. Our findings suggest that astrocytes throughout the entorhinal cortex of AD patients gradually accumulate A beta 42-positive material and that the amount of this material correlates positively with the extent of local AD pathology. A beta 42-positive material within astrocytes appears to be of neuronal origin, most likely accumulated via phagocytosis of local degenerated dendrites and synapses, especially in the cortical molecular layer. The co-localization of neuron-specific proteins, alpha 7 nicotinic acetylcholine receptor and choline acetyltransferase, in A beta 42-burdened, activated astrocytes supports this possibility. Our results also suggest that some astrocytes containing A beta 42-positive deposits undergo lysis, resulting in the formation of astrocyte-derived amyloid plaques in the cortical molecular layer in brain regions showing moderate to advanced AD pathology. These astrocytic plaques can be distinguished from those arising from neuronal lysis by virtue of their smaller size, their nearly exclusive localization in the subpial portion of the molecular layer of the cerebrocortex, and by their intense glial fibrillary acidic protein immunoreactivity. Overall, A beta 42 accumulation and the selective lysis of A beta 42-burdened neurons and astrocytes appear to make a major contribution to the observed amyloid plaques in AD brains.     

Hematopoietic prostaglandin D synthase and DP1 receptor are selectively upregulated in microglia and astrocytes within senile plaques from human patients and in a mouse model of Alzheimer disease

Prostaglandin (PG) D2 is produced in activated microglia by the action of hematopoietic PGD synthase (HPGDS) and plays important roles in neuroinflammation. Because the fact that neuroinflammation accelerates progression of Alzheimer disease (AD) has been documented, we investigated whether PGD2 is also involved in the pathology of AD. Here, we report that the level of the mRNA of the receptor for PGD2 (DP1) was increased in AD brains compared with the level in non-AD brains. Immunocytochemical analysis showed HPGDS expression to be localized in the microglia surrounding senile plaques. In situ hybridization studies revealed that DP1 mRNA was specifically localized in microglia and reactive astrocytes within senile plaques of AD brains. In the brain of Tg2576 mice, a model of AD, HPGDS and DP1 proteins were mainly localized immunocytochemically in microglia and astrocytes in the plaques, and the levels of their mRNAs increased in parallel with amyloid beta deposition. These results indicate that PGD2 may act as a mediator of plaque-associated inflammation in AD brain and may explain the pharmacologic mechanisms underlying the favorable response of patients with AD to nonsteroidal anti-inflammatory drugs.     

Aberrant expression of NOS isoforms in Alzheimer's disease is structurally related to nitrotyrosine formation

Various isoforms of the nitric oxide (NO) producing enzyme nitric oxide synthase (NOS) are elevated in Alzheimer's disease (AD) indicating a critical role for NO in the pathomechanism. NO can react with superoxide to generate peroxynitrite, a process referred to as oxidative stress, which is likely to play a role in AD. Peroxynitrite in turn, nitrates tyrosine residues to form nitrotyrosine which can be identified immunohistochemically. To study the potential structural link between the increased synthesis of NO and the deposition of nitrotyrosine in AD, we analyzed the expression of neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) in AD and control brain, and compared the localization with the distribution of nitrotyrosine. Nitrotyrosine was detected in neurons, astrocytes and blood vessels in AD cases. Aberrant expression of nNOS in cortical pyramidal cells was highly co-localized with nitrotyrosine. Furthermore, iNOS and eNOS were highly expressed in astrocytes in AD. In addition, double immunolabeling studies revealed that in these glial cells iNOS and eNOS are co-localized with nitrotyrosine. Therefore, it is suggested that increased expression of all NOS isoforms in astrocytes and neurons contributes to the synthesis of peroxynitrite which leads to generation of nitrotyrosine. In view of the wide range of isoform-specific NOS inhibitors, the determination of the most responsible isoform of NOS for the formation of peroxynitrite in AD could be of therapeutic importance in the treatment of Alzheimer's disease.     

Microglial reactivity to beta-amyloid is modulated by astrocytes and proinflammatory factors

The brains of Alzheimer's disease (AD) patients present activated glial cells, amyloid plaques and dystrophic neurites. The core of amyloid plaques is composed of aggregated amyloid peptide (Abeta), a peptide known to activate glial cells and to have neurotoxic effects. We evaluated the capability of glial cells to mediate Abeta(1-42) cytotoxicity in hippocampal cultures. Conditioned media obtained from microglial cultures exposed to Abeta induced apoptosis of hippocampal cells. This pro-apoptotic effect was not observed in hippocampal cultures exposed to conditioned media obtained from mixed glial (astrocytes and microglia) cultures that had been exposed to Abeta. Microglia exposed to Abeta responded with reactive morphological changes, induction of iNOS, elevated nitric oxide production and decreased reductive metabolism. All these responses were attenuated by the presence of astrocytes. This astrocyte modulation was however, not observed when glial cells were exposed to proinflammatory factors (LPS+Interferon-gamma) alone or in combination with Abeta. Our results suggest that astrocytes and proinflammatory molecules are determining factors in the response of microglia to Abeta.     

Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer's disease brains

J. Satoh, H. Tabunoki, T. Ishida, Y. Saito and K. Arima (2013) Neuropathology and Applied Neurobiology 39, 109–120 Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer's disease brains Aims: RGMa is a repulsive guidance molecule that induces the collapse of axonal growth cones by interacting with the receptor neogenin in the central nervous system during development. It remains unknown whether RGMa plays a role in the neurodegenerative process of Alzheimer's disease (AD). We hypothesize that RGMa, if it is concentrated on amyloid plaques, might contribute to a regenerative failure of degenerating axons in AD brains. Methods: By immunohistochemistry, we studied RGMa and neogenin (NEO1) expression in the frontal cortex and the hippocampus of 6 AD and 12 control cases. The levels of RGMa expression were determined by qRT‐PCR and Western blot in cultured human astrocytes following exposure to cytokines and amyloid beta (Aβ) peptides. Results: In AD brains, an intense RGMa immunoreactivity was identified on amyloid plaques and in the glial scar. In the control brains, the glial scar and vascular foot processes of astrocytes expressed RGMa immunoreactivity, while oligodendrocytes and microglia were negative for RGMa. In AD brains, a small subset of amyloid plaques expressed a weak NEO1 immunoreactivity, while some reactive astrocytes in both AD and control brains showed an intense NEO1 immunoreactivity. In human astrocytes, transforming growth factor beta‐1 (TGFβ₁), Aβ_1–40 or Aβ_1–42 markedly elevated the levels of RGMa, and TGFβ₁ also increased its own levels. Coimmunoprecipitation analysis validated the molecular interaction between RGMa and the C‐terminal fragment β of amyloid beta precursor protein (APP). Furthermore, recombinant RGMa protein interacted with amyloid plaques in situ. Conclusions: RGMa, produced by TGFβ‐activated astrocytes and accumulated in amyloid plaques and the glial scar, could contribute to the regenerative failure of degenerating axons in AD brains.     

Cell cycle related signaling in neuro2a cells proceeds via the receptor for advanced glycation end products

Summary Re-expression of cell cycle related genes such as cyclin-dependent kinases (cdk), cyclins, or cdk inhibitors in differentiated neurons in Alzheimer’s disease (AD) is rooted in aberrant mitogenic signaling. Since microglia and astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque components or factors secreted from plaque-activated glia induce mitogenic signaling in neurons. Mitogenic compounds might be S100B, overexpressed by activated astrocytes, or advanced glycation end products (AGEs), a component of plaques. Both S100B and AGEs may interact with the multiligand receptor for AGEs (RAGE) and trigger for the activation of the p42/44 mitogen-activated protein kinase (p42/44 MAPK), whether they also count for cell cycle related signaling in neurons remains unresolved. By immunohistochemical staining, we confirmed that cyclin D₁ positive neurons are surrounded by AGE deposits, demonstrating the potential relevance in vivo. For exploring the mitogenic signal cascade, we used Neuro2a cells overexpressing human full-length RAGE (FL-RAGE) or the cytosolic deletion mutant (Δ-RAGE). In both cell lines, S100B and AGEs induced the production of reactive oxygen species but not in a RAGE-dependent manner. By contrast, in FL-RAGE cells but not in Δ-RAGE cells S100B and AGEs activate p42/44 MAPK, augment cyclin D₁/cdk4 protein and RNA levels and the transition into the S-phase. Moreover, in FL-RAGE cells, decreased protein levels of the cdk inhibitor p16 were observed, and the p42/44 MAPK inhibitor UO126 prevented AGE and S100B stimulated cyclin D₁ expression and hindered cells to enter the S-phase. Our results demonstrate that S100B and AGE may serve as mitogenic sources for the stimulation of neurons to progress through the cell cycle whereby signaling proceeds via RAGE → p42/44 MAPK → cyclin D₁/cdk4.     

Neuregulin-1 and erbB4 immunoreactivity is associated with neuritic plaques in Alzheimer disease brain and in a transgenic model of Alzheimer disease

Neuregulin-1 (NRG-1) regulates developmental neuronal survival and synaptogenesis, astrocytic differentiation, and microglial activation. Given these NRG-1 actions, we hypothesized that the synaptic loss, gliosis, inflammation, and neuronal death occurring in Alzheimer disease (AD) is associated with altered expression of NRG-1 and its receptors (the erbB2, erbB3, and erbB4 membrane tyrosine kinases). We examined the expression and distribution of NRG-1 and the erbB kinases in the hippocampus of AD patients and cognitively normal controls and in transgenic mice that coexpress AD-associated mutations of the beta amyloid precursor protein (APP(K670N,M671L)) and presenilin-1 (PS1(M146L)). In the hippocampi of both control humans and wild type mice, NRG-1 and the 3 erbB receptors are expressed in distinct cellular compartments of hippocampal neurons. All 4 molecules are associated with neuronal cell bodies, but only NRG-1, erbB2, and erbB4 are present in synapse-rich regions. In AD and in the doubly transgenic mouse, erbB4 is expressed by reactive astrocytes and microglia surrounding neuritic plaques. In AD brains, microglia and, to a lesser extent, dystrophic neurites, also upregulate NRG-1 in neuritic plaques, suggesting that autocrine and/or paracrine interactions regulate NRG-1 action within these lesions. NRG-1 and erbB4, as well as erbB2, are similarly associated with neuritic plaques in the doubly transgenic mice. Thus, in AD the hippocampal distribution of NRG-1 and erbB4 is altered. The similarities between the alterations in the expression of NRG-1 and its receptors in human AD and in APP(K670N;M671L)/PS1(M146L) mutant mice suggests that this animal model may be very informative in deciphering the potential role of these molecules in AD.     

Down‐regulation of MET in hippocampal neurons of Alzheimer's disease brains

We found that mRNA of MET, the receptor of hepatocyte growth factor (HGF), is significantly decreased in the hippocampus of Alzheimer's disease (AD) patients. Therefore, we tried to determine the cellular component‐dependent changes of MET expressions. In this study, we examined cellular distribution of MET in the cerebral neocortices and hippocampi of 12 AD and 11 normal controls without brain diseases. In normal brains, MET immunoreactivity was observed in the neuronal perikarya and a subpopulation of astrocytes mainly in the subpial layer and white matter. In AD brains, we found marked decline of MET in hippocampal pyramidal neurons and granule cells of dentate gyrus. The decline was more obvious in the pyramidal neurons of the hippocampi than that in the neocortical neurons. In addition, we found strong MET immunostaining in reactive astrocytes, including those near senile plaques. Given the neurotrophic effects of the HGF/MET pathway, this decline may adversely affect neuronal survival in AD cases. Because it has been reported that HGF is also up‐regulated around senile plaques, β‐amyloid deposition might be associated with astrocytosis through the HGF signaling pathway.     

Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosis brains

In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.     

Peroxiredoxin 6 in human brain: molecular forms,cellular distribution and association with Alzheimer’s disease pathology

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient's with Alzheimer's disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a "halo" around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to A beta peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.     

Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer's disease

The cellular distribution of malondialdehyde (MDA) was assessed immunohistochemically in brain specimens from young and normal elderly subjects as well as patients with Alzheimer's disease (AD). MDA was increased in the cytoplasm of neurons and astrocytes in both normal aging and AD, but was rarely detected in normal young subjects. By electron microscopic immunohistochemistry, neuronal MDA formed cap-like linear deposits associated with lipofuscin, while glial MDA deposits surrounded the vacuoles in a linear distribution. In the hippocampus, neuronal and glial MDA deposition was marked in the CA4 region but mild in CA1. By examination of serial sections stained with anti-MDA and antibodies against an advanced glycation end product, N(epsilon)-(carboxymethyl)lysine (CML), neuronal and glial MDA deposition was colocalized with CML in AD, but only neuronal MDA was colocalized with CML in normal aged brains. Glial MDA, although abundant in the aged brain, typically was not colocalized with CML. In AD cases, MDA was colocalized with tau protein in CA2 hippocampal neurons; such colocalization was rare in CA1. MDA also was stained in cores of senile plaques. Thus, while both MDA and CML accumulate under oxidative stress, CML accumulation is largely limited to neurons, in normal aging, while MDA also accumulates in glia. In AD, both MDA and CML are deposited in both astrocytes and neurons.     

Phosphorylation of MARCKS in Alzheimer disease brains

Activation of the amyloid beta-protein precursor, secretary pathway through alpha-secretase has been reported to increase the secretion of neuroprotective amyloid precursor protein and preclude the formation of amyloid beta-protein. Activation of protein kinase C has been shown to accelerate this secretory pathway. These results prompted us to focus on a potential links between protein kinase C and the amyloid beta-protein-related pathology of Alzheimer disease (AD). Although protein kinase C is reported to occur in senile plaques, its catalytic activity has not been investigated. As the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) has been used as a marker for activation of protein kinase C in vivo, we examined its phosphorylation in brain tissues obtained from seven AD patients and five non-demented subjects using an antibody that specifically recognized MARCKS phosphorylated by protein kinase C. Phosphorylation of MARCKS in cortical neurons in AD brains was weaker than that in control brains. Interestingly, however, phosphorylation of MARCKS was detected in microglia and dystrophic neurites within neuritic plaques, a mature form of amyloid beta-protein deposits. These results suggest that protein kinase C alteration is associated with AD pathology and that protein kinase C is activated in microglia and dystrophic neurites by amyloid beta-protein in AD brains.     

Iron deposits in multiple sclerosis and Alzheimer''s disease brains

Iron may contribute to the pathogenesis of neurological diseases by promoting oxidative damage. The localization of iron in multiple sclerosis (MS) and Alzheimer's disease (AD) brains was investigated to further the understanding of its pathogenic role in these disease states. Earlier studies, utilizing a standard Perls' stain, yielded conflicting reports regarding the distribution of iron deposits in MS brains, and a previous study on AD brains utilized a diaminobenzidine (DAB) enhanced version of this stain. In the present study, a modified version of the DAB-enhanced stain was used; it utilizes sodium borohydride, proteinase K, Triton X-100 and xylenes to increase the accessibility of tissue iron to histochemical reagents. This modified method can reveal iron deposits that are missed by the Perls' or DAB-enhanced Perls' stains. In addition to its normal deposition in oligodendrocytes and myelin, iron was detected in reactive microglia, ameboid microglia and macrophages in MS brains. In AD brains, three types of plaques were stained: dense core, clear core and amorphous plaques. Punctate staining was also observed in neurons in the corticies of AD brains. The structure accounting for punctate labeling may be damaged mitochondria, lipofuscin or amyloid deposits. Dense core plaques, clear plaques and punctate labeling were not detected in the previous AD study which utilized only the DAB-enhanced Perls' stain. The labeling of these additional structures illustrates the benefit of the modified method. In summary, the localization of iron deposition in MS and AD brains indicates potential sites where iron could promote oxidative damage in these disease states.     

Regression stage senile plaques in the natural course of Alzheimer's disease

Resolution process of cerebroparenchymal amyloid beta-protein (Abeta) deposition has become of increasing interest in the light of recent advance in the Abeta-vaccination therapy for Alzheimer's disease (AD). However, the neuropathological features of degraded and disappearing senile plaque remain poorly characterized, especially in the natural course of the disease. To clarify the natural removal processes of Abeta burden in the brain with AD, we devised a triple-step staining method: Bodian for dystrophic neurites, anti-glial fibrillary acidic protein for astrocytes, and anti-Abeta. We thus examined 24 autopsied AD brains. A novel form of senile plaques, termed 'remnant plaques', was identified. Remnant plaques were characterized by mesh-like astroglial fibrils within the entire plaque part, Abeta deposit debris exhibiting weak Abeta immunoreactivity, and only a few slender dystrophic neurites. In remnant plaques, amyloid burden was apparently decreased. The density of remnant plaques increased significantly with disease duration. Dual-labelling immunohistochemistry revealed many Abeta-immunoreactive granules in astrocytes and a modest number in microglia, both of which accumulated in senile plaques. We consider amyloid deposits of diffuse and neuritic plaques to be shredded by astrocytic processes from the marginal zone of plaques, and to gradually disintegrate into smaller compartments. Cerebroparenchymal Abeta deposits undergo degradation. After a long-standing resolution process, diffuse and neuritic plaques may finally proceed to remnant plaques. Astrocytes are actively engaged in the natural Abeta clearance mechanism in advanced stage AD brains, which may provide clues for developing new therapeutic strategies for AD.