Toshiki Uchihara

ApoE immunoreactivity and microglial cells in Alzheimer's disease brain.

The spatial relationship of apolipoprotein E (apoE)-like immunoreactivity (IR) to amyloid beta-peptide (A beta), astrocytes and microglial cells in the brain of Alzheimers disease was studied by double immunolabelling. Diffuse apoE-like IR was seen in A beta diffuse deposits, and markedly increased in the core of classic senile plaques. Microglial cells, sometimes immunoreactive for apoE, were frequent in areas of apoE-like IR, where they often grouped into clusters in the core of apoE-labelled senile plaques. Although astrocytic processes were seen within these senile plaques, the cell bodies were always at a distance from the core. None of these astrocytes expressed apoE-like IR. Microglial cells, some of them immunoreactive for apoE, were seen in the center of apoE-labelled senile plaques. These data suggest that microglial cells play a more significant role than astrocytes in apoE deposition in senile plaques of Alzheimer disease.

Increased Expression of Neuronal Apolipoprotein E in Human Brain With Cerebral Infarction

Background and Purpose— Cellular origin of apolipoprotein E (ApoE) in the human brain and its roles in physiological and pathological conditions remain to be clarified. Methods— Immunolocalization of ApoE was investigated in a series of autopsied human brains with or without infarction. ApoE expression was also estimated on immunoblot on protein extracts from autopsied brains and a cultured neuroblastoma cell line of human origin (GOTO) subjected to an oxidative stress induced by exposure to hydrogen peroxide (0.2 mmol/L). Results— In addition to astrocytes and microglia, neurons and degenerated axons in and around the ischemic foci contained ApoE-like immunoreactivity, which was more intense in recent ischemic foci. Immunoblot demonstrated an increase in expression of ApoE in brain extracts from ischemic lesion, and this increase was also pronounced in the cultured neuroblastoma cell line after the stress. Conclusions— Accumulation of ApoE in neurons in and around ischemic foci of the human brain is related to an increase in ApoE synthesis in neurons, as seen in cultured neuronal cells after oxidative stress. Intrinsic regenerative activity of neuron in reaction to external insults may be related to this increase in ApoE of neuronal origin.

14-3-3 proteins and zeta isoform containing neurofibrillary tangles in patients with Alzheimer’s disease

Immunolocalization of 14-3-3 proteins in Alzheimer’s disease (AD) brains was investigated using isoform-specific antibodies. Weak granular immunoreactivity of 14-3-3 proteins was found in neuronal cytoplasm in control subjects and AD brains. Both intracellular and extracellular neurofibrillary tangles (NFTs), as well as neuropil thread-like structures, were immunopositive for 14-3-3 proteins. This was corroborated by triple-fluorolabeling method visualizing paired helical filament (PHF) tau and 14-3-3 epitopes in relation to fibrillary state detected by thiazin red. Pretangle neurons (positive for PHF-tau without fibrillary structure detected by thiazin red) only contained fine granular immunoreactivity (IR) of 14-3-3, which was similarly found in unaffected neurons. Granular cytoplasmic IR of 14-3-3 proteins in pretangle neurons was not colocalized to granular tau-like IR, which suggests that participation of 14-3-3 proteins in NFT formation was restricted to its later stages. Its zeta isoform was most prominent in these NFTs, suggesting that this isoform is a major component involved in the formation of NFTs. In contrast, IR of epsilon isoform was found in the neuropil of the hippocampus and that of sigma isoform was localized to granule cells of the dentate gyrus in AD brains, as seen in the age-matched controls. Expression of 14-3-3 proteins were found to be highly variable and dependent on their isoforms, regions and cell types. Molecular, as well as topographical, dissection of 14-3-3 proteins will provide us with an improved understanding of this molecule in normal and pathological conditions.

PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases

In polyglutamine diseases, accumulation in the nucleus of mutant proteins induces the formation of neuronal intranuclear inclusions (NIIs). The nucleus is compartmentalized into structural and functional domains, which are involved in NII formation. Promyelocytic leukemia protein (PML), a major component of nuclear bodies, and mSin3A, a component of the transcription co-repressor complex, were used to investigate how the intranuclear domains/sites relate to NII formation in SCA2, SCA3, SCA7, SCA17 and DRPLA brains. We demonstrate that the size of PML-positive intranuclear structures was larger in pathological brains than in control ones and that these structures contained mutant proteins. PML colocalized only with small NIIs, which maintained the ring-like structure of normal nuclear bodies. Enlarged ring-like PML-positive structures, devoid of mutant proteins, were also found and might represent structures where mutant polyglutamine proteins have been successfully processed. These data suggest that NIIs originate from nuclear bodies, where mutant proteins accumulate for degradation.

Widespread immunoreactivity of presenilin in neurons of normal and Alzheimer's disease brains: double-labeling immunohistochemical study.

Abstract The immunolocalization of presenilin in human brain was studied using two antibodies raised against different portions of presenilin 1 (S182) protein. A granular staining was found in the cytoplasm of neurons in cortical layers III and V. One of the antibodies, also reactive to presenilin 2 (E5-1) protein, additionally stained dendrites and axons. This was seen in normal brains as well as in brains affected by Alzheimer’s disease. Less prominent immunolabeling was noted in some senile plaques. No relationship to neurofibrillary tangles was found in double-labeling experiments combined with anti-paired helical filament-tau antibody (AT8). The widespread expression of presenilin in normal brain suggests a physiological role of the protein.

Heterogeneity of Nigral and Cortical Lewy Bodies Differentiated by Amplified Triple-Labeling for Alpha-Synuclein, Ubiquitin, and Thiazin Red

Alpha-synuclein(alpha-S) and ubiquitin(Ub) are constituents of the Lewy bodies (LBs), composed of fibrillary structures. To clarify morphological heterogeneity of LBs, we looked for localization of these epitopes in relation to fibrillary structure possibly detectable by a fluorochrome, thiazin red (TR). On the sections of the substantia nigra (SN) and the cingulate gyrus (CG) obtained from Parkinsons disease brains, double amplification by CARD fluorescent immunohistochemistry with anti-alpha-S monoclonal (LB509) and anti-Ub polyclonal antibodies was performed, followed by staining with TR. These triple-labeled images were captured by a confocal laser microscope and subsequently stained with Campbell-Switzer method, a silver staining specific for LBs. Staining profiles of LBs were different between those in the SN and in the CG. Immunolabeling either with the anti-alpha-S or anti-Ub antibody was diffuse without halo structure in LBs of CG. In addition to this diffuse staining, a lot of LBs of SN exhibited a halo structure immunopositive for alpha-S and Ub, probably representing later stages of LB evolution. Irrespective of the presence of this halo structure, the TR signal was always concentrated in the center of LBs, as the silver-stained material was, suggesting that fibrillary components in the central portion of LBs undergo some conformational changes detectable by TR and the silver-staining. This technique reveals different epitopes in relation to LB evolution in vivo. Heterogeneity in staining profile of LBs, as clarified by this method, may represent evolutional changes of LBs, related to conformational states of their constituents.

Constant involvement of the Betz cells and pyramidal tract in amyotrophic lateral sclerosis with dementia: a clinicopathological study of eight autopsy cases

Abstract. We investigated clinicopathologically pyramidal signs, including hyperreflexia, Babinski sign, and spasticity, and the involvement of the primary motor cortex and pyramidal tract, in eight Japanese autopsy cases of amyotrophic lateral sclerosis (ALS) with dementia. Pyramidal signs were observed in seven (88%) of the eight autopsy cases. Hyperreflexia and Babinski sign were evident in seven (88%) and three (38%) patients, respectively, but spasticity was not observed in any of the eight patients. Loss of Betz cells in the primary motor cortex was evident in the seven cases in which this structure was examined. Astrocytosis in the fifth layer of the primary motor cortex was noticed in three cases. In all eight cases, involvement of the pyramidal tract was obvious in the medulla oblongata, but no involvement of the pyramidal tract was found in the midbrain. Involvement of the pyramidal tract in the spinal cord, particularly of large myelinated fibers, was observed in all six cases in which the spinal cord was examined. In ALS with dementia, pyramidal signs were shown to be present more frequently than previously believed, and the clinicopathological correlation between pyramidal signs and involvement of the pyramidal tract was obvious. Constant involvement of Betz cells and the pyramidal tract in ALS with dementia has not been reported. Our clinicopathological findings may make a contribution to the understanding of the clinicopathological hallmarks of this disorder. Furthermore, we believe that this study will also contribute to the elucidation of the nosological status of ALS with dementia.

Pick body disease and Pick syndrome

Diagnostic criteria for Picks disease have been criticized from many different viewpoints. This confusion is mainly derived from the ambiguity of this term ‘Picks disease’ (PD), which may imply either purely histological findings, such as Pick body (PB), or a characteristic clinical syndrome that could occur even in the absence of PB. This taxonomic confusion will be circumvented by introducing the diagnostic term ‘Pick body disease’ to designate patients with the characteristic argyrophilic inclusions purely on histological grounds. In parallel, employment of ‘Pick syndrome’ to describe the time‐honored clinical features may be more convenient and less confusing than PD because PD implies either the presence of PB or the clinical features, two aspects not necessarily linked to each other. Three‐dimensional reconstruction of PB confirmed that tau‐like immunoreactivity was accentuated at their periphery, as was recognized with the Bodian method. Preferential affinity of three‐repeat tau pathology, as seen in Pick body disease, to the Bodian over the Gallyas method is distinct from the reversed affinity (the Gallyas over the Bodian method) of four‐repeat tau pathology, as seen in corticobasal degeneration and in argyrophilic grains. This preference of silver staining is compatible with the mixed three‐ and four‐repeat tau pathology, as seen in NFT of the Alzheimers type, which are stained with both the Bodian and Gallyas staining. This will provide a practical basis on which to differentiate these disorders based on their distinctive tau species and possible relation of tau species to staining profile on these silver methods.

Triple Immunofluorolabeling with Two Rabbit Polyclonal Antibodies and a Mouse Monoclonal Antibody Allowing Three-dimensional Analysis of Cotton Wool Plaques in Alzheimer Disease

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Aβ42 or anti-Aβ40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Aβ40 or anti-Ap42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 μm were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Aβ40 was localized to core-like structures, mainly in layers I–III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I–VI, were labeled with anti-Aβ42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Aβ deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.

Immunolocalization of 14-3-3 isoforms in brains with Pick body disease

Immunolocalization of 14-3-3 protein isoforms in relation to Pick bodies in Pick body disease (PBD) brains was investigated. Weakly granular immunoreactivity of 14-3-3 proteins was found in neurons in control subjects and in Pick body disease brains. In addition to this granular immunoreactivity, many Pick bodies were immunopositive for 14-3-3 proteins as confirmed with double-immunofluorescence with an anti-PHF tau (AT8) and anti-14-3-3 that recognizes all its isoforms (common). When probed with isoform-specific antibodies, Pick bodies were positive for beta, gamma, epsilon, eta, tau, and zeta isoform and exhibited immunostaining pattern similar to that observed with the anti-14-3-3 proteins (common). In addition, immunoreactivity of sigma isoform, so far considered to be exclusively extraneuronal, was unexpectedly found in Pick bodies, normal hippocampal neurons and brain homogenate from age-matched controls. Although localization of 14-3-3 proteins in Pick bodies suggests their involvement in Pick body formation, their role may be variable dependent on the isoforms differently expressed in different area in the brain.