Takahiko Umahara

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.

Association Between ApoE Phenotypes and Telomere Erosion in Alzheimer’s Disease

Although several reports suggest that Alzheimers disease (AD) is associated with shortened telomere length, the clinical relevance of this has not yet been fully elucidated. This study was conducted to clarify the correlation of telomere length with clinical characteristics and ApoE phenotypes in 74 AD patients. Telomere length was determined from genomic DNA extracted from whole blood by quantitative real-time polymerase chain reaction. We found no significant difference in telomere length between the AD and non-dementia elderly control (n = 35) groups. Furthermore, no significant correlation was found among telomere length and the severity of cognitive decline and disease duration, age, or gender difference. However, telomere length was significantly shorter in AD patients with the ApoE4 homozygote than in those with the ApoE4 heterozygote (p < .001) and noncarriers (p < .001). These findings suggest that shortened telomere length may be associated with the ApoE4 homozygote in AD patients.

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.

Telomere length shortening in patients with dementia with Lewy bodies

Background and purpose:  Shortened telomere length has been considered to be associated with various age‐related diseases, especially in dementia such as Alzheimer’s disease and vascular dementia. However, changes in telomere length in dementia with Lewy bodies (DLB) remain unclear. To elucidate these changes, we set out to determine telomere length in peripheral leukocytes as well as the level of urinary 8‐hydroxy‐deoxyguanosine (8‐OHdG) as a marker of oxidative stress in DLB.

Intranuclear localization and isoform-dependent translocation of 14-3-3 proteins in human brain with infarction

Immunolocalization of 14-3-3 proteins in human brains with infarction was investigated using isoform-specific antibodies. Neurons around acute or subacute ischemic foci exhibited an enhanced immunoreactivity for 14-3-3 proteins either in the cytoplasm (especially for its sigma isoform) or in the nucleus (especially for its beta isoform), and sometimes in both. 14-3-3-like immunoreactivity was evaluated in each neuron, which enabled us to identify into three patterns: intense cytoplasmic staining with or without nuclear staining; a predominant nuclear staining with weak cytoplasmic staining; and an exclusive nuclear staining without cytoplamic staining. Quantification of 1500 neurons in relation to the severity of ischemia estimated by the relative distance from ischemic foci clarified that nuclear immunoreactivity for 14-3-3 proteins was more frequent in neurons near the ischemic core. Although the cytoplasm of astrocytes was similarly positive for the sigma and the epsilon isoform, their nuclei were only immunopositive for the gamma isoform. In the cerebral white matter with ischemia, axonal swelling and some nuclei of oligodendrocytes were positive for the zeta isoform. Isoform-specific translocation of 14-3-3 proteins into nuclei is a cellular reaction to ischemic stress that may be related to survival of neurons and their protection against cell death.

Intranuclear immunolocalization of 14-3-3 protein isoforms in brains with spinocerebellar ataxia type 1

Immunolocalization of 14-3-3 protein isoforms, one of the interacters with ataxin 1, was investigated in spinocerebellar ataxia type 1 (SCA 1) brains using isoform-specific antibodies. Samples from the pons and from the cerebellum of four SCA1 cases and three controls were studied. The intensity of the immunoreactivity (IR) and its subcellular topography were analyzed. In control subjects, granular immunoreactivity for an epitope common to all known isoforms of 14-3-3 proteins (14-3-3 COM) found in the cytoplasm of some pontine and dentate nucleus neurons was weak. It was observed in some Purkinje cells, while its intensity varied. Many nuclei of those neurons and Purkinje cells of SCA1 were intensely immunopositive for 14-3-3 COM, while it was less in their cytoplasm. Expanded polyglutamine epitope was colocalized to 14-3-3 COM epitope in some pontine neurons, sometimes accumulated in intranuclear inclusion-like structures. This findings support previous reports that 14-3-3 proteins stabilize mutant ataxin 1 in nucleus and possibly lead to neurodegeneration. However, nuclear localization of 14-3-3 proteins in SCA1 brains was dependent on its isoforms, i.e. pontine neurons intensely positive for beta, Purkinje cells for tau and dentate nucleus neurons for both, while all of those neurons were consistently positive for zeta isoform, although sigma isoform tended to be located in the cytoplasm. Nuclear accumulation and isoform- and region-dependent subcellular localizations of 14-3-3 proteins may be related to SCA1 pathology, which exhibits marked regional variability.

14-3-3 proteins and spinocerebellar ataxia type 1: from molecular interaction to human neuropathology.

This mini-review focuses on the possible relevance of 14-3-3 proteins in spinocerebellar ataxia type 1 (SCA1). 14-3-3 proteins are mainly localized in the synapses and neuronal cytoplasm, and seven isoforms have been identified in mammals. This family of proteins was initially identified as adaptor proteins which bind to phosphoserine-containing motifs. Binding motifs and potential functions of 14-3-3 proteins are now recognized to have a wide range of functional relevance. SCA1 is an autosomal-dominant neurodegenerative disorder and is linked to polyglutamine expansion (ataxin-1 protein). The Zoghbi and Orr group showed direct interaction of 14-3-3 proteins with ataxin-1 where nuclear recruitment of the ataxin-1 protein is dependent on its phosphorylation. This targeted binding of 14-3-3 protein to phosphorylated ataxin-1 to stabilize ataxin-1 in cellular models was corroborated by our double-labeling study for expanded polyglutamine and 14-3-3 proteins which demonstrated colocalization of these two epitopes in the neuronal nuclei in human autopsied brains with SCA1. Ataxin-1/14-3-3 protein interaction is a new potential target for therapeutic intervention in the treatment of SCA1.

Differential expression of 14-3-3 protein isoforms in developing rat hippocampus, cortex, rostral migratory stream, olfactory bulb, and white matter.

We investigated the differential immunoexpression of 14-3-3 proteins according to their 7 isoforms during the postnatal development of rat brains, primarily in the hippocampus, cortex, rostral migratory stream (RMS), olfactory bulb, and white matter. Wistar rats at different developmental stages, on postnatal days 2 (P2), P7, P14, P21 and P100 were obtained, and were incubated with each type of anti-14-3-3 isoform antibody. 14-3-3 common (COM)-like immunoreactivity (IR) which represents an epitope shared among the 7 isoforms was initially expressed in the olfactory bulb on P2. This IR was partially expressed in the dentate granule cells and hippocampal pyramidal neurons from P7, and increased during development. These chronological changes were similar to those obtained with beta, gamma, and eta isoforms. Epsilon isoform-like IR was initially identified in the cell body of cortical neurons and glia-like cells on P2. After P7, the IR was more intense in the neuropil of the cortex. This epsilon isoform-like IR was markedly accentuated in the stratum lucidum of the hippocampus after P7, where hippocampal mossy fibers terminate, functioning as a giant synapse. This suggests that epsilon isoforms may be associated with synaptogenesis of the hippocampal mossy fibers. Sigma isoform-like IR was observed in the nuclei of external plexiform layer cells of the olfactory bulb from P2 to P21, in the nuclei of the hippocampal pyramidal and dentate granule cells after P7 and in the nuclei of RMS cells after P7. Zeta and tau isoform-like IRs were mainly identified in the white matter and in oligodendroglial cells from P7 to P21. Different immunolocalizations of the 7 isoforms suggest that 14-3-3 protein isoforms are individually associated with neuronal development and synaptogenesis during postnatal formation of the rat brain.

Differential expression of oxidized/native lipoprotein(a) and plasminogen in human carotid and cerebral artery plaques.

OBJECTIVE Lipoprotein(a) [Lp(a)] is a risk factor for stroke, as has recently been further confirmed by meta analysis, and consists of low-density lipoprotein and apolipoprotein(a), which shares a significant amino acid homology with plasminogen. Oxidized Lp(a) [Ox-Lp(a)] is a more pathogenic species of Lp(a). A monoclonal antibody, specific to Ox-Lp(a), distinguishes immunolocalization of Ox-Lp(a) from that of Lp(a) and that of plasminogen which carries highly homologous domains. It is worth examining their possibly differential immunolocalizations around atherosclerotic lesions in human carotid and cerebral arteries. METHODS AND RESULTS Stage-related differences in immunolocalization of Ox-Lp(a), native Lp(a), and plasminogen were investigated in various atherosclerotic lesions of the human carotid (obtained from 13 patients undergoing carotid endarterectomy) and cerebral arteries (from 11 cadavers). Native Lp(a) was seen in the fibrous cap, extracellular matrix, endothelial cells, and subendothelial layer. Unorganized mural thrombi were positive for plasminogen, but not Lp(a). In contrast, fibrin deposits in thickened intima were positive for Lp(a), but not plasminogen. Ox-Lp(a)-like immunoreactivity was seen in endothelial cells in the early stage of atherosclerosis. Ox-Lp(a) deposition was more abundant in synthetic phase vascular smooth muscle cells (VSMC) than in contractile phase VSMC. CONCLUSION We demonstrated differential immunoexpression patterns between native Lp(a) and plasminogen, and suggested that Lp(a)-plasminogen interaction may play a part in differential mechanisms in all atherosclerotic lesions of human carotid and cerebral arteries. The preferential presence of Ox-Lp(a) seen in endothelial cells suggests initial dysfunction of endothelial cells in atherosclerosis. The relative abundance of Ox-Lp(a) in synthetic phase VSMC is associated with their phenotypic changes during the progression of atherosclerosis.

Isoform-dependent immunolocalization of 14-3-3 proteins in developing rat cerebellum

We investigated the expression of 14-3-3 protein and its 7 isoforms during postnatal development of rat cerebellum with immunoblot and immunohistochemistry with isoform-specific antibodies. The relative amounts of total 14-3-3 protein, probed by an antibody (14-3-3 COM) recognizing a sequence shared among its isoforms, exhibited no significant changes from postnatal day 2 (P2) to P100. 14-3-3 COM-like immunoreactivity (IR), initially in the apical portion of Purkinje cells at P2, extended to Purkinje cell bodies at P14 and to their dendrites (P100) with increasing intensity. Molecular layer (after P7) and cerebellar nucleus neurons (after P14) were also immunolabeled with this antibody. These chronological changes were shared with those obtained with beta, gamma, and eta isoforms. In contrast, epsilon isoform-like IR was initially identified in processes of radial and Bergmann glia at P2 prior to its appearance in the molecular layer at P7 with subsequent intensification also in Purkinje cells after P14. Zeta and tau isoform-like IR was identified in the white matter and/or in oligodendroglial cells. The sigma isoform was the only isoform exhibiting a significant quantitative change with a peak at P14. Immunolocalization of sigma isoform was initially restricted in several cells in Purkinje cell layer at P2 and shifted to nuclei of external and internal granule cells and Purkinje cells after P14, whereas its immunolabeling was markedly weaker at P100. Different immunolocalizations of the 7 isoforms suggest that 14-3-3 protein isoforms individually associate with the neuronal and glial proliferation, differentiation, migration and development during postnatal formation of rat cerebellum.