Granulovacuolar Degeneration Granules Research Articles

Alzheimer’s disease‐related necroptotic pathology: An exclusive presence of the necrosome in granulovacuolar degeneration inclusions in human and transgenic mouse brains

AbstractBackgroundAlzheimer’s disease (AD) is characterized by pathological processes such as β‐amyloid (Aβ) plaques, neurofibrillary tangles (NFT), cytoplasmic granulovacuolar degeneration (GVD) lesions and neuronal loss that underpin cognitiveand behavioral alterations. Necroptosis, a programmed form of cell death defined by the assembly of the necrosome complex composed of phosphorylated proteins (pRIPK1, pRIPK3 and pMLKL), has recently been shown to be involved in AD.MethodTo determine the neuropathological characteristics and distribution pattern of the necrosome components, we performed a comprehensive immunohistochemical analysis of brains of AD, pathologically defined preclinical AD and non‐AD control cases. A similar analysis was carried out to investigate necroptosis‐related proteins in transgenic mouse models of Aβ and Tau pathology.ResultsWe identified all three activated necrosome components (pRIPK1, pRIPK3 and pMLKL) in GVD inclusionswithin neurons of the hippocampal subfields CA1, CA2/3, CA4, dentate gyrus, subiculum, entorhinal cortex, temporal cortex, hypothalamus, amygdala and frontal cortex of symptomatic and preclinical AD patients. The three necrosome proteins colocalized with standard GVD markers, such as pTDP‐43 and CK1δ, and similarly to these markers detected GVD lesions. Necrosome‐associated GVD neurons inversely correlated with neuronal density in the early affected hippocampus and in the late affected frontal cortex. Necroptosis‐specific GVD lesions showed strong positive associations with AD‐defining parameters, showing the strongest correlation and partial colocalization with Tau pathology. Moreover, we analyzed brain tissues of transgenic mice carrying mutated APP, Tau or both transgenes. We found morphologically comparable GVD lesions positive for the necroptosis components in the subiculum, hippocampus and entorhinal cortex of 12‐month‐old APP23xTAU58 mice. We confirmed mouse necrosome‐positive GVD inclusions by CK1δ immunoreactivity.ConclusionThe presence of the necrosome in GVD inclusions points to a specific form of necroptosis in AD that can be modelled in APPxTAU transgenic mice, possibly indicating Tau and/or Aβ as drivers of this pathology. Necrosome‐positive GVD granules represent a potential protective system sequestering the necrosome complex, which could interfere with or delay necroptosis‐related neuronal death.

Increased prevalence of granulovacuolar degeneration in C9orf72 mutation.

Granulovacuolar degeneration (GVD) is usually found in Alzheimer's disease (AD) cases or in elderly individuals. Its severity correlates positively with the density of neurofibrillary tangles (NFTs). Mechanisms underlying GVD formation are unknown. We assessed the prevalence and distribution of GVD in cases with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Consecutively autopsied cases with FTLD/ALS-TDP and C9orf72 mutations (FTLD/ALS-C9; N = 29), cases with FTLD/ALS-TDP without C9orf72 mutations (FTLD/ALS-nonC9; N = 46), and age-matched healthy controls (N = 40) were studied. The prevalence of GVD was significantly higher in the FTLD/ALS-C9 cases (26/29 cases) than in the FTLD/ALS-nonC9 cases (15/46 cases; Fisher exact test; p < 2×10-6) or in the control group (12/40 individuals; p < 1×10-6). Average Braak stages and ages of death were not significantly different among the groups. The CA2 sector was most frequently affected in the FTLD/ALS-C9 group, whereas the CA1/subiculum was the most vulnerable area in the other groups. Extension of GVD correlated with the clinical duration of the disease in the FTLD/ALS-C9 cases but not in the FTLD/ALS-nonC9 cases. The GVD-containing neurons frequently had dipeptide repeat (DPR) protein inclusions. GVD granules labeled with antibodies directed against charged multivesicular body protein 2B or casein kinase 1δ were attached to DPR inclusions within GVD. Our results suggest that development of GVD and DPR inclusions is related to common pathogenic mechanisms and that GVD is not only associated with NFTs seen in AD cases or aging individuals.

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.

Vacuole-creating protein in neurodegenerative diseases in humans

Vacuole-creating protein (VCP) is a member of the ATPases associated with diverse cellular activities and is a putative sensor protein for degenerative proteins. Immunohistochemical examinations demonstrated that VCP was observed in ubiquitin-positive intraneuronal inclusions in motor neuron disease with dementia, ballooned neurons in Creutzfeldt–Jakob disease, dystrophic neurites of senile plaque in Alzheimer's disease, and Lewy and Marinesco bodies and Lewy neurites in Parkinson's disease, while granules of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer's disease were not positively stained for VCP. These results indicate that VCP reacts with abnormal or misfolded proteins and plays a role in accelerating the process of degeneration and cell death. The elucidation of an association between VCP and these degenerative proteins will provide an important clue for understanding common mechanisms underlying neurodegenerative diseases.

Caspase-cleaved amyloid precursor protein and activated caspase-3 are co-localized in the granules of granulovacuolar degeneration in Alzheimer's disease and Down's syndrome brain.

Granulovacuolar degeneration (GVD) is a diagnostic neuropathological feature of Alzheimer's disease (AD). In some neurons, apoptosis has been hypothesized to be a primary mechanism causing neuronal cell death in AD. In this study we investigated CA1 neurons with GVD in AD and Down's syndrome (DS) brain. We demonstrated that activated caspase-3 and a caspase-cleaved cleavage product of the amyloid precursor protein (cAPP) are co-localized in GVD granules, and that these same cells often show nuclear DNA damage. In contrast, activated caspase-8 is present in the cytoplasm but not within the granules of GVD neurons. A caspase-cleavage product of fodrin that accumulates in many AD and DS neurons is not present in GVD granules. These data support a role for the activation of apoptotic mechanisms in selective compartments exhibiting GVD.

The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.

Glycogen synthase kinase-3beta (GSK-3beta) is a physiological kinase for tau and is a candidate protein kinase involved in the hyperphosphorylation of tau present in paired helical filament (PHF)-tau of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). GSK-3beta is also a key element of several signaling cascades (including cell death cascades). We have investigated the immunocytochemical localization of GSK-3 immunoreactivity in AD. Neurons exhibiting strongly GSK-3-immunoreactive granules were observed in AD, with a much higher frequency than in control subjects. This immunoreactivity was found to co-localize with the granulovacuolar degeneration (GVD) and to be associated with the granules of the granulovacuolar bodies. The GVD granules showed a strong GSK-3alpha and GSK-3beta immunoreactivity, and this immunoreactivity was abolished by preabsorption with recombinant GSK-3. In addition, the GVD immunoreactivity was observed with an antibody against the tyrosine-phosphorylated and active form of GSK-3. Some granules of the granulovacuolar degeneration were also intensely labeled with an antibody specific for tau isoforms containing insert 1 (exon 2) and with antibodies specific for tau phosphorylated on Ser262 and for tau phosphorylated on Thr212/Ser214, two phosphorylation sites generated in vitro by GSK-3alpha and beta. GSK-3beta was expressed in neurons containing NFT but only a small proportion of intracellular NFT were observed to be GSK-3beta immunoreactive. Immunoblotting analysis of fractions enriched in PHF-tau did not reveal any GSK-3beta immunoreactivity in these fractions, indicating that GSK-3beta was only loosely associated to NFT. These results suggest that neurons developing GVD sequester an active, potentially deleterious, form of GSK-3 in this compartment and that increased GSK-3 immunoreactivity in a subset of neurons quantitatively differentiates normal aging from AD.

Activation of Caspase-3 in Single Neurons and Autophagic Granules of Granulovacuolar Degeneration in Alzheimer's Disease: Evidence for Apoptotic Cell Death

Neuronal loss is prominent in Alzheimer's disease (AD), and its mechanisms remain unresolved. Apoptotic cell death has been implicated on the basis of studies demonstrating DNA fragmentation and an up-regulation of proapoptotic proteins in the AD brain. However, DNA fragmentation in neurons is too frequent to account for the continuous neuronal loss in a degenerative disease extending over many years. Furthermore, the typical apoptotic morphology has not been convincingly documented in AD neurons with fragmented DNA. We report the detection of the activated form of caspase-3, the central effector enzyme of the apoptotic cascade, in AD and Down's syndrome (DS) brain using an affinity-purified antiserum. In AD and DS, single neurons with apoptotic morphology showed cytoplasmic immunoreactivity for activated caspase-3, whereas no neurons were labeled in age-matched controls. Apoptotic neurons were identified at an approximate frequency of 1 in 1100 to 5000 neurons in the cases examined. Furthermore, caspase-3 immunoreactivity was detected in granules of granulovacuolar degeneration. Our results provide direct evidence for apoptotic neuronal death in AD with a frequency compatible with the progression of neuronal degeneration in this chronic disease and identify autophagic vacuoles of granulovacuolar degeneration as possible means for the protective segregation of early apoptotic alterations in the neuronal cytoplasm.