Shiro Sugihara

Microglial activation in early stages of amyloid β protein deposition

Abstract The present study was undertaken to investigate the relationship of microglial activation to amyloid β protein (Aβ) deposition, particularly at the early stage. Using single and double immunostaining methods with a panel of microglia markers and antibodies against Aβ and amyloid β protein precursor (APP), we examined the cerebrum and cerebella of both Alzheimer’s disease (AD) and non-demented subjects obtained at autopsy. In non-demented, middle-aged subjects that had small amounts of cerebral Aβ deposits, approximately 70% of the diffuse plaques contained ramified microglia. However, no evidence of microglial activation was found in diffuse plaques in any of the non-demented subjects. Dual immunostaining of sections of cerebral cortex using antibodies against Aβ and major histocompatibility complex class II antigen showed that in AD subjects, approximately 20% of total diffuse plaques contained a few, activated microglia. Most of these plaques were defined as a transitional form between diffuse and primitive plaques. Both primitive and classic plaques in the cerebral cortex of AD subjects consistently contained clusters of activated microglia. Subpial Aβ deposits without neuritic changes lacked microglial activation. In the cerebellum, all of the diffuse plaques lacked microglial activation, and activated microglia in the compact plaques were not as hypertrophic as those in cerebral primitive/classic plaques. Our findings indicate that microglial reactions are absent in the early stages of Aβ deposition, and it occurs during the transition from diffuse to primitive plaques, when amounts of Aβ deposits and the degree of neuritic changes increase.

Diffuse plaques associated with astroglial amyloid β protein, possibly showing a disappearing stage of senile plaques

Abstract To clarify whether senile plaques disappear, we examined amyloid β protein (Aβ) deposits in non-demented subjects, and found novel diffuse plaques associated with astroglial Aβ. Formalin-fixed paraffin-embedded sections from cortical areas were immunolabeled with a panel of Aβ antibodies, and astroglial and microglial markers. Cerebral Aβ deposition was primarily found as diffuse plaques (DP) in these subjects. A subset of DP was associated with clusters of intensely Aβ-positive small granules. The clusters, which were located just adjacent to astroglial nucleus, had the characteristics of lipofuscin granules and, therefore, were quite different from “small stellate deposits”. Substantial amounts of Aβ-positive granules were found inside astrocytes by dual labeling of Aβ and glial fibrillary acid protein, and the majority of astroglial Aβ immunoreactivity was located on lipofuscin granules. Aβ-positive granules lacked immunoreactivity with antisera for the N-terminal region of Aβ. These peculiar DP showed a much weaker staining than ordinary DP. The DP associated with astroglial Aβ were found in about one third of the subjects, although the density varied widely among individuals. From these findings, we propose that DP, which are associated with the N-terminal truncated Aβ in astrocytes, represent the disappearing stage of senile plaques.

Effect of Apolipoprotein E Allele ε4 on the Initial Phase of Amyloid β-Protein Accumulation in the Human Brain

Deposition of amyloid β-protein (Aβ), a hallmark of Alzheimers disease, occurs to some extent in the brains of most elderly individuals. We sought to learn when Aβ deposition begins and how deposition is affected by apolipoprotein E allele e4, a strong risk factor for late-onset Alzheimers disease. Using an improved extraction protocol and specific enzyme-linked immunosorbent assay, we quantified the levels of Aβ40 and Aβ42 in the insoluble fractions of brains from 105 autopsy cases, aged 22 to 81 years at death, who showed no signs of dementia. Aβ40 and Aβ42 were detected in the insoluble fractions from all of the brains examined; low levels were even found in the brains of patients as young as 20 to 30 years of age. The incidence of significant Aβ accumulation increased age-dependently, with Aβ42 levels beginning to rise steeply in some patients in their late 40s, accompanied by much smaller increases in Aβ40 levels. The presence of the apolipoprotein E e4 allele was found to significantly enhance the accumulation of Aβ42 and, to a lesser extent, that of Aβ40. These findings strongly suggest that the presence of e4 allele results in an earlier onset of Aβ42 accumulation in the brain.

Appearance of Sodium Dodecyl Sulfate-Stable Amyloid β-Protein (Aβ) Dimer in the Cortex During Aging

We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid β-protein (Aβ) by enzyme immunoassay (EIA) provided prominent signals at 6∼8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Aβ dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Aβ, capture SDS-dissociable Aβ but not SDS-stable Aβ dimer. Thus, all cortical specimens in which the levels of Aβ were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6∼8 kd, but not SDS-dissociable Aβ monomer at ∼4 kd, as judged from the blot. This Aβ dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the Aβ dimer, and (ii) until 12 hours postmortem, such SDS-stable Aβ dimer is detected only faintly in PDAPP transgenic mice. The presence of Aβ dimer in the cortex may characterize the accumulation of Aβ in the human brain, which takes much longer than that in PDAPP transgenic mice.

Presence of apolipoprotein E on extracellular neurofibrillary tangles and on meningeal blood vessels precedes the Alzheimer β-amyloid deposition

The localization of apolipoprotein E (ApoE) has been examined immunohistochemically in the autopsied brains of middle-aged and old-aged control subjects, with and without amyloid β protein (Aβ) deposits, and of Alzheimers disease patients. Senile plaques were consistently labeled with ApoE antiserum even in the very early stage of senile plaque formation seen in the fifth decade. In the cerebellar molecular layer, small dots of ApoE immunoreactivity, which were prominent in the Alzheimers disease subjects, were observed in addition to immunoreactivity in diffuse plaques. ApoE antisera labeled all of the extracellular neurofibrillary tangles (NFT), whereas only a small minority of extracellular NFT were positive for Aβ. A punctate pattern of ApoE immunoreactivity was seen at the media of the meningeal vessels lacking amyloid, when senile plaques were present in the nearby cortex. In the early stage of amyloid angiopathy, the distribution of ApoE immunoreactivity was much more extensive than that of Aβ positivity. These findings suggest that ApoE accumulates in the early stage of senile plaque formation and, furthermore, that ApoE accumulation precedes Aβ deposition in extracellular NFT and amyloid angiopathy.

Accumulation of Amyloid β-Protein in the Low-Density Membrane Domain Accurately Reflects the Extent of β-Amyloid Deposition in the Brain

To learn more about the process of amyloid β-protein (Aβ) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Aβ content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Aβ. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Aβ deposits in those cases in which Aβ accumulated to significant levels. Aβ42 levels in the low-density membrane domain were proportional to the extent of total Aβ42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Aβ in a similar manner to aging humans, Aβ42 accumulation in the low-density membrane domain also increased as Aβ deposition progressed with aging. These observations indicate that the Aβ42 associated with low-density membrane domains is tightly coupled with the process of extracellular Aβ deposition.

The immunophenotype of perivascular cells in the human brain

The immunophenotype of perivascular cells (PC) in temporal lobe tissues obtained at autopsy in 48 patients (aged 41–88 years) was characterized using light and electron microscopic immunocytochemistry with a variety of antibodies. In all cases studied, PC bearing CD11 c (Ki‐M1 P) and CD68 (KP1) were distributed throughout the temporal cortex. In addition to Ki‐M1P and KP1, the monoclonal antibodies against major histocompatibility complex (MHC) class II antigen (Ag) (LN‐3, CR3.43), anti‐leucocyte common antigen (LCA), LN‐5, Mac 387 were all found in PC with variable immunoreactivity. In contrast, LN‐1 and OPD4 were not found in PC, although the former showed nearly constant staining of resting microglia. Semiquantitative analysis disclosed differences in the numbers of cells labeled with the markers in the 21 normal brains (KI‐M1P > KP1 » LCA, LN‐3, LN‐5 » Mac 387). Ultrastructurally, immunoreactivity for Ki‐M1P, KP1, and LN‐3 was observed in PC with cytoplasm containing dense lysosomal bodies. In brains from patients with Alzheimers type dementia, PC were seen in the wall of β‐amyloid protein‐positive small vessels. However, there was no definite alteration of anti‐genicity in PC from AD brains compared with those from normal brains. The immunophenotype of PC was similar to that of macrophages, which were observed in the perivascular spaces and the leptomeninges in some normal and diseased brains. In contrast with PC, however, macrophages showed high incidence of labeling for some mac‐rophage markers LN‐5 and Mac 387. These findings demonstrate that PC may be a normal constituent of the adult human brain with a variable expression of monocyte/ macrophages markers and MHC class II Ag and that PC could be distinguished from resting microglia by their morphology and by their immunophenotype.

Immunohistochemical analysis of COOH-termini of amyloid beta protein (Aβ) using end-specific antisera for Aβ40 and Aβ42 in Alzheimer's disease and normal aging

We examined by immunohistochemicistry the carboxyl (C)-terminus extent of the amyloid beta protein (Aβ) that constitutes senile plaques and amyloid angiopathy in the brains of non-demented and Alzheimers disease (AD) subjects. We developed two antisera, which selectively recognized free C-termini of Aβ: BC40 for Aβ40; and BC42 for Aβ42. BC42 labeled various types of senile plaques as well as reference Aβ antiserum, whereas only some parts of the senile plaques were positive with BC40: i.e., all of the plaque cores and some diffuse and primitive plaques. In the brains of non-demented middle-aged subjects, a majority of BC42-positive diffuse plaques were also positive with BC40, but with less intensity than that shown with BC42. The ratio of BC40-negative plaques increased with increasing plaque density. Amyloid in the meningeal vessels showed much greater immnnoreactivity with BC40 than with BC42. Some extracellular neurofibrillary tangles were positive with both BC40 and BC42, although most of them and a...

Cerebral β amyloid deposition in patients with malignant neoplasms: its prevalence with aging and effects of radiation therapy on vascular amyloid

We examined immunohistochemically 123 autopsy brains from patients aged between 30 to 59, who died as a result of malignant neoplasms. Using antiserum to amyloid β protein (Aβ), we found that cerebral Aβ deposits began in the subjects fifth decade; its prevalence was 0%, 9.8% and 21.5% in the fourth, fifth and sixth decades, respectively. The major form of Aβ deposition was diffuse-type plaques, although one third of the brains with Aβ deposition showed amyloid angiopathy. Subpial Aβ deposition is frequently associated with amyloid angiopathy. The prevalence of cerebral Aβ deposits was about two times higher in the patients who had received brain radiation therapy (27.8%) compared to non-radiated patients (14.8%). Amyloid angiopathy was much more prominent (P<0.05) with radiation therapy (22.2%) than without (8.0%). We found that cerebral Aβ deposition is dependent on aging, even in patients with malignant tumors and at beginning in their forties, and that brain radiation therapy is a possible risk factor of Aβ deposition, especially in the form of amyloid angiopathy.

Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain.

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the α‐ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimers disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH‐SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD‐deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse‐label experiment revealed that the loss of the subunits is a post‐translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.