Granulovacuolar Degeneration Research Articles

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

BackgroundGranulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimer's disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders.MethodsAn autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized.ResultsThe number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs.ConclusionsOur data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation.

Stages of granulovacuolar degeneration: their relation to Alzheimer’s disease and chronic stress response

Granulovacuolar degeneration (GVD) is characterized by the presence of vacuolar cytoplasmic lesions in nerve cells of the medial temporal lobe. These changes occur in older non-diseased individuals as well as in patients with Alzheimer's disease (AD), progressive supranuclear palsy (PSP), Pick's, sporadic Parkinson's (PD), and Guam diseases. We stained representative paraffin sections from all parts of the brain with anti-pTDP43, anti-CK1δ or anti-CK1ε from 14 non-demented elderly, 19 AD, 17 non-AD tauopathy, 9 sporadic PD, and 5 TDP43-proteinopathy [amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)] cases. Our results showed five stages of GVD based on its distribution pattern: GVD began in the hippocampal subfields CA1, CA2, and the subiculum. In a second stage, entorhinal cortex, and CA4 neurons exhibited GVD. Additional neurons were involved in the temporal neocortex in stage 3, whereas the affection of the amygdala and/or the hypothalamus marked stage 4. A fifth and final stage was characterized by additional GVD in the cingulate cortex and occasionally in the frontal and parietal cortices as well as in the oral raphe and pedunculopontine tegmental nuclei. The GVD stages correlated with neurofibrillary tangle stages, Consortium to Establish a Registry for AD (CERAD) scores for neuritic plaque pathology, amyloid β-protein deposition phases, cerebral amyloid angiopathy stages, and clinical dementia rating (CDR) scores. No associations were seen between GVD stage and the presence of non-AD tauopathies, PD, ALS, or FTLD cases. In conclusion, GVD affects neurons in a hierarchical sequence that allows the distinction of five stages. The topographic distribution of GVD restricted to regions involved in response to chronic stress could indicate a link between GVD and chronically stressful influences. Moreover, the association of the GVD stages with those of AD-related pathology but not with other neurodegenerative disorders points to a possible role of GVD and the response to chronic stress in the pathogenesis of AD.

Stages of granulovacuolar degeneration and its relation to Alzheimer's disease

Stages of granulovacuolar degeneration and its relation to Alzheimer's disease

Granulovacuolar degeneration bodies of Alzheimer's disease resemble late‐stage autophagic organelles

Granulovacuolar degeneration involves the accumulation of large, double membrane‐bound bodies within certain neurons during the course of Alzheimer's disease. Because of the two‐layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. To test this hypothesis, spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for granulovacuolar degeneration bodies (GVBs), was investigated in hippocampal sections prepared from post mortem Alzheimer's disease cases using double‐label confocal fluorescence microscopy. Results revealed that GVBs colocalized most strongly with LAMP1 (lysosome‐associated membrane protein 1) and CHMP2B (charged multivesicular body protein 2B), but weakly with lysosomal marker cathepsin D or early‐stage autophagy markers LC3 and p62. The resultant immunohistochemical signature suggests that GVBs do contain late‐stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. The data further suggest that failure to complete autolysosome formation may be an important correlate of GVB accumulation. This work was supported in part by the National Institute on Aging (AG14452, JK).

Granulovacuolar degeneration (GVD) bodies of Alzheimer's disease (AD) resemble late-stage autophagic organelles

Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurones during the course of Alzheimer's disease (AD) and other adult-onset dementias. Because of the two-layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. The aim of this study was to test this hypothesis, and determine the approximate stage at which the pathway stalls in AD. Spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for granulovacuolar degeneration (GVD) bodies, was evaluated in hippocampal sections prepared from post mortem Braak stage IV and V AD cases using double-label confocal fluorescence microscopy. GVD bodies colocalized weakly with early-stage autophagy markers LC3 and p62, but strongly with late-stage marker lysosome-associated membrane protein 1 (LAMP1), which decorated their surrounding membranes. GVD bodies also colocalized strongly with charged multivesicular body protein 2B (CHMP2B), which colocalized with the core granule, but less strongly with lysosomal marker cathepsin D. The resultant immunohistochemical signature suggests that granulovacuolar degeneration bodies (GVBs) do contain late-stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. The data further suggest that failure to complete autolysosome formation may be an important correlate of GVB accumulation.

Histopathological and Immunohistochemical Comparison of the Brain of Human Patients with Alzheimer’s Disease and the Brain of Aged Dogs with Cognitive Dysfunction

Alzheimer's disease (AD) is the most common progressive form of dementia in aged people. Microscopical changes in the brains of AD patients include the formation of senile plaques (SPs), neurofibrillary tangles (NFTs) and granulovacuolar degeneration and the deposition of amyloid-beta (Aβ). Aged dogs are known to suffer from cognitive dysfunction and this state is associated with deposition of Aβ in the brain. The aim of the present study was to investigate tau phosphorylation of neurons and astrocytes in the brain of aged dogs with progressive cognitive impairment. Changes in the brain of aged dogs with cognitive dysfunction were compared with those in the brain of patients with AD of Braak stage V. Immunohistochemically, Aβ deposition, phosphorylated tau Ser396 (p-tau Ser396) and ubiquitin were observed in the parietal cortex and hippocampus of aged dogs with cognitive dysfunction. Astrocytes with expression of p-tau Ser396 and neurons with co-localization of p-tau Ser396 and ubiquitin were observed. Expression of p-tau Ser396 and accumulation of ubiquitin were significantly increased in the parietal cortex and dorsal part of the hippocampus of the brain of aged dogs when compared with expression of these molecules in human AD.

Involvement of endoplasmic reticulum stress defined by activated unfolded protein response in multiple system atrophy

Multiple system atrophy (MSA) and Parkinson's disease (PD) are classified as synucleinopathies that exhibit α-synuclein deposition in the central nervous system. Recently, activation of the unfolded protein response (UPR), which is a cellular stress response triggered by endoplasmic reticulum (ER) stress, was reported in PD and involvement of ER stress was indicated for this disease. To elucidate whether ER stress is also implicated in the pathology of MSA, we performed a series of immunohistochemical studies using MSA brain sections. Here, we showed the presence of an activated UPR response in oligodendroglia of postmortem MSA brains. The UPR protein-positive structures were observed in lesions where glial cytoplasmic inclusions (GCIs) appeared and colocalized highly in cells showing oligodendrocytic characteristics in the presence of α-synuclein inclusions. The UPR protein-positive structures appeared as granular shapes that are morphologically similar to granulovacuolar degeneration (GVD) and colocalized with GVD marker proteins. Double immunohistochemistry demonstrated that some of the activated UPR protein-positive structures were localized in oligodendrocytes that contained GCIs with faint α-synuclein labeling, without ubiquitination, and showing a strong correlation with the relocation of the tubulin polymerization-promoting protein (TPPP/p25α). These findings suggest that activation of the UPR may be induced at the early stage of the disease process, thus playing a pivotal role in the pathology of MSA.

Lipid peroxidation markers are associated with granulovacuolar degeneration but not lipofuscin formation

Lipid peroxidation markers are associated with granulovacuolar degeneration but not lipofuscin formation

Immunopositivity for ESCRT-III subunit CHMP2B in granulovacuolar degeneration of neurons in the Alzheimer's disease hippocampus

Endosomal sorting complex required for transport (ESCRT)-III subunit charged multivesicular body protein 2B (CHMP2B) is involved in the degradation of proteins in the endocytic and autophagic pathways. Mutations in the CHMP2B gene are reportedly associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) characterised by accumulation of ubiquitinated protein aggregates in affected neurons, suggesting a relationship between protein accumulation and efficient autophagic degradation. This study investigated CHMP2B immunoreactivity in the hippocampus of patients with Alzheimer's disease (AD), revealing intense labeling of intraneuronal dot-like structures by antibody to CHMP2B. Since the morphological characteristics of these granular structures were compatible with those of granulovacuolar degeneration (GVD), a hallmark of AD pathology, immunohistochemical study using anti-CHMP2B antibody was performed using AD and control brain sections to investigate whether this antibody can be used as a GVD label. The number and percentage of hippocampal neurons with CHMP2B-positive granules were higher in AD cases and CHMP2B-positive granules corresponded to GVD. Anti-CHMP2B antibody detected a single 28-kDa band on Western blotting using control and AD specimens. This antibody clearly and intensely detected GVD over the hippocampus and entorhinal and transentorhinal cortices. These findings suggest that researchers will be able to use CHMP2B as a molecular label for studying GVD.

Paxillin and hydrogen peroxide-inducible clone 5 expression and distribution in control and Alzheimer disease hippocampi.

Hydrogen peroxide-inducible clone 5 (Hic-5) and paxillin are members of the Group III LIM domain protein family that localize to both cell nuclei and focal adhesions and link integrin-mediated signaling to the actin cytoskeleton. Prior in vitro studies have implicated paxillin in beta-amyloid-induced cell death, but little is known about the expression and function of Hic-5 and paxillin in the brain. We performed a blinded retrospective cross-sectional study of Hic-5 and paxillin expression in the hippocampi of Alzheimer disease (AD) and control subjects using immunohistochemistry and laser scanning confocal microscopy. The analysis included assessment of the expression of phosphorylated isoforms of paxillin that reflect activation of distinct signaling pathways. We found changes in the subcellular distribution of Hic-5, paxillin, and specific phosphorylated isoforms of paxillin in the AD brains. The Hic-5 and phosphorylated isoforms of paxillin colocalized with neurofibrillary tangles. Paxillin was predominantly found in reactive astrocytes in the AD hippocampi, and activated paxillin was also detected in granulovacuolar degeneration bodies in AD. These data indicate that these important scaffolding proteins that link various intracellular signaling pathways to the extracellular matrix are modified and have altered subcellular distribution in AD.

Widespread distribution of reticulon-3 in various neurodegenerative diseases

Reticulons are a group of membrane-bound proteins involved in diverse cellular functions, and are suggested to act as inhibitors of β-secretase enzyme 1 (BACE1) activity that cleaves amyloid precursor protein. Reticulons are known to accumulate in the dystrophic neurites of Alzheimer's disease (AD), and studies have suggested that alterations in reticulons, such as increased aggregation, impair BACE1 binding, increasing amyloid-β production, and facilitating reticulon deposition in dystrophic neurites. To further characterize the cellular distribution of reticulon, we examined reticulon-3 expression in cases of AD, Parkinson's disease, and diffuse Lewy body disease. A more widespread cellular distribution of reticulon-3 was noted than in previous reports, including deposits in dystrophic neurites, neuropil threads, granulovacuolar degeneration, glial cells, morphologically normal neurons in both hippocampal pyramidal cell layer and cerebral neocortex, and specifically neurofibrillary tangles and Lewy bodies. These results are compatible with reticulon alterations as nonspecific downstream stress responses, consistent with its expression during periods of endoplasmic reticulum stress. This emphasizes the increasing recognition that much of the AD pathological spectrum represents a response to the disease rather than cause, and emphasizes the importance of examining upstream processes, such as oxidative stress, that have functional effects prior to the onset of structural alterations.

Neuropathology of Alzheimer's Disease

Alois Alzheimer first pointed out that the disease which would later bear his name has a distinct and recognizable neuropathological substrate. Since then, much has been added to our understanding of the pathological lesions associated with the condition. The 2 primary cardinal lesions associated with Alzheimer's disease are the neurofibrillary tangle and the senile plaque. The neurofibrillary tangle consists of abnormal accumulations of abnormally phosphorylated tau within the perikaryal cytoplasm of certain neurons. The senile plaque consists of a central core of beta-amyloid, a 4-kD peptide, surrounded by abnormally configured neuronal processes or neurites. Other neuropathological lesions are encountered in cases of Alzheimer's disease, but the disease is defined and recognized by these 2 cardinal lesions. Other lesions include poorly understood changes such as granulovacuolar degeneration and eosinophilic rodlike bodies (Hirano bodies). The loss of synaptic components is a change that clearly has a significant impact on cognitive function and represents another important morphological alteration. It is important to recognize that distinguishing between Alzheimer's disease, especially in its early stages, and normal aging may be very difficult, particularly if one is examining the brains of patients who died at an advanced old age. It is also noted that instances of pure forms of Alzheimer's disease, in the absence of other coexistent brain disease processes, such as infarctions or Parkinson's disease-related lesions, are relatively uncommon, and this must be taken into account by researchers who employ postmortem brain tissues for research.

Phosphorylation-dependent TDP-43 antibody detects intraneuronal dot-like structures showing morphological characters of granulovacuolar degeneration

TAR-DNA-binding protein 43 (TDP-43) was considered to be a disease-specific component of ubiquitin-positive and tau-negative inclusions in the brains of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis (ALS). However, this protein also accumulates abnormally in neurons in other neurodegenerative diseases, including Alzheimer's disease (AD). Although the role of TDP-43 deposition in these diseases is not clear, abnormal phosphorylation of the protein is suggested to be a critical step in disease pathogenesis. In this study, we generated a new phosphorylation-dependent TDP-43 antibody and examined AD brain sections from temporal lobes, including the hippocampus and temporal neocortex, by immunohistochemistry. The antibody, called A2, specifically recognized phosphorylated TDP-43 in western blotting using ALS and AD specimens, detecting a strong 45kDa band and several shorter fragments at around 25kDa with smears. Immunohistochemistry demonstrated neuronal cytoplasmic inclusions in AD brain sections without staining nuclei that were normal physiological TDP-43 localization sites. These results were consistent with previous reports. However, intraneuronal dot-like structures were also intensely labeled by immunohistochemistry. These structures were observed in all the AD brain sections examined and also occurred in sections from the brains of aged subjects without AD pathologies. The morphological and immunohistochemical characteristics of these granular structures were compatible with those of granulovacuolar degeneration (GVD). The A2 antibody clearly and intensely detected granular structures distributed over the hippocampus, subiculum, parahippocampus and temporal neocortex. Thus, immunohistochemistry using phosphorylation-dependent TDP-43 antibodies would be a new useful tool for identifying GVD.

The Unfolded Protein Response Is Activated in Pretangle Neurons in Alzheimer's Disease Hippocampus

Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2alpha, and inositol-requiring enzyme 1alpha was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3beta in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis.

Cytomorphologic manifestations of Alzheimer's disease using brain squash smears: An autopsy study with histology–cytology correlation

Alzheimer's disease (AD) is the most common form of dementia. The cardinal histopathologic features include senile plaques (SPs) and neurofibrillary tangles (NFTs), and in addition, granulovacuolar degeneration (GVD) and Hirano bodies (HBs) are frequently observed in the hippocampus. We studied hippocampal cytological features of AD, compared with controls. Hippocampal squash smears were prepared from 26 autopsy brains and stained with three different solutions, including Papanicolaou stain (Pap-s). The smears were evaluated for the aforementioned four structures and gliosis, and their frequency was compared between AD (n = 15) and control (n = 11) groups. Hippocampal smears of all AD cases revealed NFTs and gliosis. NFTs were light gray with thick flame-like structures on Pap-s. GVD was identified in the majority of AD cases and was most easily observed on Pap-s. SPs were difficult to identify and were seen only in AD cases. HBs were rarely identified as long light eosinophilic hyaline structures on Pap-s. Cytological findings of hippocampi reflect the characteristic histological features of AD with the exception of HBs, which are difficult to identify on smears. NFTs and gliosis, and SPs seem to be sensitive and specific cytologic markers in hippocampal smears for AD, respectively.

Molecular network analysis suggests aberrant CREB-mediated gene regulation in the Alzheimer disease hippocampus.

The pathogenesis of Alzheimer disease (AD) involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs) and 559 incipient AD-related genes (IADGs) identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004). By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB) as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify the in silico observations in vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB) antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD) in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.

Molecular Network Analysis Suggests Aberrant CREB-Mediated Gene Regulation in the Alzheimer Disease Hippocampus

The pathogenesis of Alzheimer disease (AD) involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs) and 559 incipient AD-related genes (IADGs) identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004). By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB) as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify thein silicoobservationsin vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB) antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD) in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.

Protective Effect of a Potent Antioxidant, Pomegranate Juice, in the Kidney of Rats with Nephrolithiasis Induced by Ethylene Glycol

We aimed to study the protective effects of pomegranate juice (PJ) on ethylene glycol (EG)-induced crystal deposition in renal tubules, renal toxicity, and inducible nitric oxide synthase (iNOS) and nuclear factor-kappaB activities in rat kidneys. Fifty-six rats were divided into four equal groups: Control, EG, EG + 50 microL PJ/d (PJ50), and EG + 100 microL PJ/d (PJ100). Rats were sacrified on days 10 and 45. Tissue sections were evaluated under light and polarized microscopy for the presence and degree of crystal deposition and toxicity in the kidneys. Crude extracts of the cortex were used to determine reduced gluthatione (GSH), nitric oxide (NO), and malondialdehyde (MDA) levels. In the EG group, crystal depositions were more evident and mild crystalization was observed in proximal tubules on day 10; severe crystalization and granulovacuolar epithelial cell degeneration were observed on day 45. There was limited or no crystal formation in the EG + PJ-given groups. There were completely normal renal and tubular structures in the control group. There was no significant difference between the four groups in serum levels of sodium, potassium, blood urea nitrogen, and creatinine in any sampling time. Hyperoxaluria, a marked increase in MDA and NO levels, and decrease of GSH were observed in the EG-given groups compared with the others. There were marked iNOS and p65 expressions in only the EG-given rats compared with control and PJ groups, immunohistochemically. This experiment shows the protective effect of PJ in the EG-induced crystal depositions in renal tubules.

The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

Targets of Caspase-6 Activity in Human Neurons and Alzheimer Disease

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and alpha-Tubulin. A neoepitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.