Granulovacuolar Degeneration Research Articles

MTOR Hyperactivation in down syndrome hippocampus appears early during development.

The mammalian target of rapamycin (mTOR) signaling pathway is a key developmental pathway involved in mechanisms underlying cellular aging and neurodegeneration. We hypothesized that its deregulation may occur during early brain development in patients with Down syndrome (DS). The expression patterns and cellular distribution of components of mTOR signaling (phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR) were investigated in developing hippocampi from controls and patients with DS and from adults with DS and Alzheimer disease-associated pathology using immunocytochemistry. In control hippocampi, only phosphorylated S6 was detected prenatally (19-41 gestational weeks); it became undetectable 2 months postnatally. Increased expression of phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR was observed in DS hippocampus compared with controls. Phosphorylated S6 and phosphorylated S6 kinase were detected prenatally and persisted throughout postnatal development. Prominent expression of mTOR components was observed in pyramidal neurons with granulovacuolar degeneration and in neurons containing neurofibrillary tangles in the hippocampi of DS subjects with Alzheimer disease pathology. These findings suggest that a dysregulated mTOR pathway may contribute to both early hippocampal developmental abnormalities and hippocampal functional impairment developing before neurodegeneration. Moreover, the expression patterns of mTOR components in adult DS hippocampus support its association with Alzheimer disease-related histopathologic changes.

A histological study of the effect of exogenous melatonin on gentamicin induced structural alterations of proximal tubules in rats

The aim of this research was to assess the reactive changes of rat proximal tubules caused by gentamicin and the effect of relatively low doses of melatonin. 48 adult male Wistar rats were distributed into six groups of equal size which all received one of the following daily intraperitoneal injections: vehicle (5% ethanol in Ringer solution) during 11 days (C); gentamicin (80 mg/kg) during 8 days (G), two groups which concomitantly received gentamicin (80 mg/kg) during 8 days and melatonin in two different test doses (5 or 20 mg/kg) during 11 days (GM1, GM2) and two groups treated only with melatonin in two different doses (5 or 20 mg/kg) during 11 days (M1, M2). Histological analysis included qualitative and semi-quantitative light microscopy analysis of proximal tubules. Exogenous melatonin had no significant effect on the microstructure, independently of dosis. The changes of proximal tubules microstructure induced by gentamicin were expressed in the form of granulovacuolar degeneration, necrosis and desquamation. The grade of proximal tubular changes was smaller in animals who besides gentamicin received melatonin. Melatonin has a dose dependent protective effect on the structural alterations of proximal tubules of the kidney induced by gentamicin.

Phosphatidylinositol‐4,5‐bisphosphate is enriched in granulovacuolar degeneration bodies and neurofibrillary tangles

AimsAmong the pathological findings in Alzheimer’s disease (AD), the temporal and spatial profiles of granulovacuolar degeneration (GVD) bodies are characteristic in that they seem to be related to those of neurofibrillary tangles (NFTs), suggesting a common mechanism underlying the pathogenesis of these structures. Flotillin-1, a marker of lipid rafts, accumulates in lysosomes of tangle-bearing neurones in AD patients. In addition, recent reports have shown that GVD bodies accumulate at the nexus of the autophagic and endocytic pathways. The aim of this study was to elucidate the distribution of the lipid component of lipid rafts, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], in AD and other neurodegenerative disorders.MethodsWe compared PtdIns(4,5)P2 immunoreactivity in the hippocampus, entorhinal cortex and neocortex of five AD cases, 17 cases of other neurodegenerative disorders and four controls. In addition, we performed double staining using markers of GVD, NFTs and lipid rafts for further characterization.ResultsImmunohistochemical analysis revealed that PtdIns(4,5)P2 was selectively enriched in GVD bodies and NFTs. Although immunoreactivity for PtdIns(4,5)P2 was also evident in NFTs composed of hyperphosphorylated tau, PtdIns(4,5)P2 was segregated from phosphorylated tau within NFTs by double immunofluorescence staining. In contrast, PtdIns(4,5)P2 colocalized with the lipid raft markers flotillin-1 and annexin 2, within GVD bodies and NFTs.ConclusionsThese results suggest that lipid raft components including PtdIns(4,5)P2 play a role in the formation of both GVD bodies and NFTs.

MARK4 and MARK3 associate with early tau phosphorylation in Alzheimer’s disease granulovacuolar degeneration bodies

BackgroundThe progression of Alzheimer’s disease (AD) is associated with an increase of phosphorylated tau in the brain. One of the earliest phosphorylated sites on tau is Ser262 that is preferentially phosphorylated by microtubule affinity regulating kinase (MARK), of which four isoforms exist. Herein we investigated the expression of MARK1-4 in the hippocampus of non-demented elderly (NDE) and AD cases.ResultsIn situ hybridization revealed a uniform, neuronal distribution of all four isoform mRNAs in NDE and AD. Immunohistochemical analyses using isoform-selective antibodies demonstrated that MARK4 in a phosphorylated form colocalizes with p-tau Ser262 in granulovacuolar degeneration bodies (GVDs) that progressively accumulate in AD. In contrast MARK4 is largely absent in the neuronal cytoplasm. MARK3 was localized to a subset of the GVD-containing neurons and also had a weak general cytoplasmic neuronal staining in both NDE and AD. These results suggest that in AD, phosphorylated MARK3 and MARK4 are sequestered and proteolysed in GVDs. MARK1 and MARK2 were absent in GVDs and exhibited relatively uniform neuronal expressions with no apparent differences between NDE and AD.ConclusionWe found that the phosphorylated and fragmented forms of MARK4 and to some extent MARK3 are present in GVDs in AD, and that this expression is highly correlated with phosphorylation of tau at Ser262. This may represent a cellular defense mechanism to remove activated MARK and p-tau Ser262 from the cytosol, thereby reducing the phosphorylating effect on tau Ser262 that appears to be a critical step for subsequent neurodegeneration.

Calpain-Mediated Hsp70.1 Cleavage in Monkey CA1 after Ischemia Induces Similar ‘Lysosomal Vesiculosis’ to Alzheimer Neurons

For the past decade evidence has gathered for the implication of lysosomes in the development of programmed cell death. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) not only as a molecular chaperone for damaged/aged proteins but also as a guardian of lysosomal integrity. Hsp70.1-mediated lysosomal stabilization is tightly regulated, but its disorder under extreme conditions results in lysosomal rupture to cause cell death. Comparing with the pathological hallmark ‘granulo-vacuolar degenerations’ in Alzheimer’s disease, the postischemic monkey neurons were carefully observed with microscope. Intriguingly, we found very similar change, and identified it as ‘lysosomal vesiculosis’ by electron microscopy. However, the exact molecular and structural impacts of the Hsp70.1 disorder upon the lysosomal membrane are hardly elucidated in the human brain because of the practical and ethical problems. Accordingly, using the monkey brain tissues after in-vivo and in-vitro oxidative stresses, we studied molecular modifications of Hsp70.1 and its counterpart bis(monoacylglycero) phosphate (BMP), because these molecules are closely related to the lysosomal membrane stability by regulating acid sphingomyelinase. It still remains unelucidated whether ischemia/reperfusion can alter composition or amount of BMP in the brain. Regardless of the brain regions studied, the normal monkey brain tissues showed calpain-mediated cleavage of Hsp70.1 after in-vitro oxidative stress. In the CA1 after in-vivo ischemia/reperfusion, we first found that docosahexaenoic and oleic acids in BMP showed a significant decrease on days 1 and 3, compared to non-ischemic controls. Since Hsp70.1- BMP binding is indispensable for activating acid sphingomyelinase and producing ceramide to stabilize lysosomal membrane, dysfunction of Hsp70.1 and BMP presumably causes storage of sphingomyelin and deficiency of ceramide at the lysosomal membrane, leading to its destabilization in the postischemic CA1 neurons. These data, combined together, suggest that lysosomal destabilization induced by calpain-mediated cleavage of carbonylated Hsp70.1 and the concomitant BMP breakdown causes ‘lysosomal vesiculosis’.

Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

BackgroundRimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.MethodsSubjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.ResultsGVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.ConclusionsThese results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.

Antimicrobial peptide β-defensin-1 expression is upregulated in Alzheimer’s brain

BackgroundThe human β-defensins (hBDs) are a highly conserved family of cationic antimicrobial and immunomodulatory peptides expressed primarily by epithelial cells in response to invasion by bacteria, fungi and some viruses. To date, the most studied members of this family of peptides are hBD-1, -2, and -3. Expression of hBD-1 and -2 has been demonstrated previously in cultured microglia and astrocytes of both mouse and human brain. Unlike inducible hBD-2 and -3, hBD-1 is constitutively expressed and is not generally upregulated by proinflammatory factors. In this study, we investigated whether hBDs, as active components of the innate immune response, are affected by pathological events in the Alzheimer’s disease (AD) brain. We assessed the expression of hBD-1, -2, and -3 in tissue obtained at autopsy from AD and age-matched control brains.MethodsFixed and frozen choroid plexus and the CA1 region of the hippocampus were obtained at autopsy from individuals diagnosed with AD, or from age-matched control brains without diagnosed neurodegenerative disease. Histopathologically diagnosed AD brain tissue was obtained for our study. Immunocytochemical analysis was performed using affinity purified polyclonal antibodies directed against hBD-1, -2, or -3. TaqMan gene expression assays were used to quantify the mRNA of hBD-1, -2, and -3 in the choroid plexus and hippocampus. Immunocytochemical detection of iron deposits was achieved using a modified Perl’s stain for redox-active iron. In vitro experiments were performed on human primary oral epithelial cells to model the human choroid plexus epithelial response to ferric chloride. Cells were then exposed to ferric chloride added to selected wells at 0, 1, or 10 mM concentrations for 24 h at 37°C. Total mRNA was isolated to quantify hBD-1 mRNA expression by RTqPCR.ResultshBD-1 peptide is apparent in astrocytes of the AD hippocampus and hippocampal neurons, notably within granulovacuolar degeneration structures (GVD). A higher level of hBD-1 was also seen in the choroid plexus of AD brain in comparison to age-matched control tissue. Increased expression of hBD-1 mRNA was observed only in the choroid plexus of the AD brain when compared to expression level in age-matched control brain. Redox-active iron was also elevated in the AD choroid plexus and in vitro addition of Fe+3Cl3 to cultured epithelial cells induced hBD-1 mRNA expression.ConclusionsOur findings suggest interplay between hBD-1 and neuroimmunological responses in AD, marked by microglial and astrocytic activation, and increased expression of the peptide within the choroid plexus and accumulation within GVD. As a constitutively expressed component of the innate immune system, we propose that hBD-1 may be of considerable importance early in the disease process. We also demonstrate that increased iron deposition in AD may contribute to the elevated expression of hBD-1 within the choroid plexus. These findings represent a potentially important etiological aspect of Alzheimer’s disease neuropathology not previously reported.

Pathology of clinical and preclinical Alzheimer’s disease

Alzheimer's disease (AD) is characterized neuropathologically by the presence of amyloid plaques, neuritic plaques, and neurofibrillary tangles (NFTs). These lesions occur not only in demented individuals with AD but also in non-demented persons. In non-demented individuals, amyloid and neuritic plaques are usually accompanied with NFTs and are considered to represent asymptomatic or preclinical AD (pre-AD) pathology. Here, we defined and characterized neuropathological differences between clinical AD, non-demented pre-AD, and non-AD control cases. Our results show that clinical AD may be defined as cases exhibiting late stages of NFT, amyloid, and neuritic plaque pathology. This is in contrast to the neuropathological changes characteristic of pre-AD, which display early stages of these lesions. Both AD and pre-AD cases often exhibit cerebral amyloid angiopathy (CAA) and granulovacuolar degeneration (GVD), and when they do, these AD-related pathologies were at early stages in pre-AD cases and at late stages in symptomatic AD. Importantly, NFTs, GVD, and CAA were also observed in non-AD cases, i.e., in cases without amyloid plaque pathology. Moreover, soluble and dispersible, high-molecular-weight amyloid β-protein (Aβ) aggregates detected by blue-native polyacrylamide gel electrophoresis were elevated in clinical AD compared to that in pre-AD and non-AD cases. Detection of NFTs, GVD, and CAA in cases without amyloid plaques, presently classified as non-AD, is consistent with the idea that NFTs, GVD, and CAA may precede amyloid plaque pathology and may represent a pre-amyloid plaque stage of pre-AD not yet considered in the current recommendations for the neuropathological diagnosis of AD. Our finding of early stages of AD-related NFT, amyloid, and GVD pathology provides a more clear definition of pre-AD cases that is in contrast to the changes in clinical AD, which is characterized by late stages of these AD-related pathologies. The observed elevation of soluble/dispersible Aβ aggregates from pre-AD compared to that in AD cases suggests that, in addition to more widespread AD-related pathologies, soluble/dispersible Aβ aggregates in the neuropil play a role in the conversion of pre-AD to clinical AD.

Lipoprotein Lipase (LPL) is Associated with Neurite Pathology and Its Levels Are Markedly Reduced in the Dentate Gyrus of Alzheimer’s Disease Brains

Lipoprotein lipase (LPL) is involved in regulation of fatty acid metabolism, and facilitates cellular uptake of lipoproteins, lipids and lipid-soluble vitamins. We evaluated LPL distribution in healthy and Alzheimer’s disease (AD) brain tissue and its relative levels in cerebrospinal fluid. LPL immunostaining is widely present in different neuronal subgroups, microglia, astrocytes and oligodendroglia throughout cerebrum, cerebellum and spinal cord. LPL immunoreactivity is also present in leptomeninges, small blood vessels, choroid plexus and ependymal cells, Schwann cells associated with cranial nerves, and in anterior and posterior pituitary. In vitro studies have shown presence of secreted LPL in conditioned media of human cortical neuronal cell line (HCN2) and neuroblastoma cells (SK-N-SH), but not in media of cultured primary human astrocytes. LPL was present in cytoplasmic and nuclear fractions of neuronal cells and astrocytes in vitro. LPL immunoreactivity strongly associates with AD-related pathology, staining diffuse plaques, dystrophic and swollen neurites, possible Hirano bodies and activated glial cells. We observed no staining associated with neurofibrillary tangles or granulovacuolar degeneration. Granule cells of the dentate gyrus and the associated synaptic network showed significantly reduced staining in AD compared to control tissue. LPL was also reduced in AD CSF samples relative to those in controls.

Elucidating Alzheimer’s disease neuronal death by the ‘calpain-cathepsin hypothesis'

Alzheimer's disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (granulo-vacuolar degenerations) represents one of the histological hallmarks of degenerating neurons in AD, a causal link between autophagy failure and neuronal death is probable. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. The aim of this study is at elucidating the underlying mechanism of AD by comparing ischemic neuronal death of monkeys and degenerative neuronal death of AD patients. Morphologic, immunoblotting, and proteomics analyses of both the ischemic monkey (Macaca fuscata, body weight of 5–10 Kg, n=20) and human AD brains were done. The ischemic monkey data in the author's laboratory were compared to the human AD patient data of the literature. The main topics are whether an age-related decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, and how genetic factors such as apolipoprotein E (APOE) and presenilin 1 can facilitate lysosomal de stabilization. Both monkey and human brains exhibited evidence of transient or continual perturbed Ca 2+ homeostasis, calpain activation, and oxidative stresses upon Hsp70.1. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product 'hydroxynonenal' that carbonylated Hsp70.1. In the monkey brain, both the carbonylation and calpain-mediated cleavage occurred at Arg469 (i.e., key site) of Hsp70.1. This caused decrease of acid sphingomyelinase activity and accumulation of lysosomal sphingomyelin. Electron microscopic data of the AD neurons showed evidence of lysosomal destabilization and multilamellar lysosomal lipidosis. Taken together, impairments of lysosomal autophagy and stabilization should be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins (calpain-cathepsin hypothesis: Yamashima T., J Neurochem. 120:477–494, 2012).

Globular glial-like inclusions in a patient with advanced Alzheimer’s disease

disturbances and memory problems. During disease evolution, apraxia and agnosia completed the clinical picture. He was globally apathetic and presented only occasionally sprouts of aggressiveness, but no prominent behavioural alterations. There were no parkinsonism and no signs of MND. He had no family history of a neurological disorder. He died due to cardiac failure. Unfixed brain weight was 1,305 g. On gross examination, frontal and temporally accentuated brain atrophy with marked ventricular enlargement and severe atrophy of amygdala and hippocampus were observed. Basal ganglia appeared atrophic showing flattening of the head of caudate nucleus (similar to patient 2 of Ahmed’s description). Substantia nigra and locus coeruleus were unremarkable. Histologically, prominent neuronal loss, astrogliosis and microglial proliferation were observed in frontal and temporal cortices, in amygdala, hippocampus and entoand transentorhinal regions. Abundant mature, diffuse and primitive βA4-amyloid plaques (DAKO, Glostrup, Denmark, clone 6F/3D) were detected (Fig. 1a). These were distributed in all cortical areas, basal ganglia, brainstem nuclei and cerebellar cortex, corresponding to a phase 5 according to Thal et al. [6] and to an age-related plaque score C according to CERAD criteria [4]. There was mild, occipitally accentuated leptomeningeal amyloid angiopathy. Furthermore, abundant hyperphosphorylated tau (Thermo-Scientific, clone AT8) immunoreactive neuropil threads, tangles and dystrophic neurites surrounding senile plaques were observed in frontal, temporal (Fig. 1b) and parietal cortex, with no involvement of primary visual cortex. Extensive tau pathology was also detected in insula, cingulum, hippocampus and parahippocampal gyrus, basal ganglia, basal nucleus of Meynert and some brainstem nuclei. In hippocampus, abundant ghost-tangles, neuronal granulovacuolar degeneration and Hirano bodies were also We read with interest the article by Ahmed and colleagues [1] entitled “Globular glial tauopathies (GGT) presenting with motor neuron disease or frontotemporal dementia: an emerging group of 4-repeat tauopathies.” The authors present clinical, neuropathological and biochemical features of two patients, one with frontotemporal dementia (FTD) and the other with motor neuron disease (MND), both sharing the presence of characteristic globular glial 4-repeat tau positive inclusions (oligodendroglial and astrocytic) with different topographical distribution. Previously, Kovacs et al. [3] described in detail seven cases showing FTLD phenotype and abundant white matter globular glial inclusions. Both authors suggest grouping previous terms such as MSTD or FTD-P-MND/PSP-CST, in one encompassing term, as this pathology seems to represent a separate entity among tauopathies. We present the post-mortem neuropathologic study of an 82-year-old man, diagnosed with possible Alzheimer’s disease at the age of 73 years due to progressive word finding

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.

Cyclin-dependent kinase 5 immunoreactivity for granulovacuolar degeneration

In addition to senile plaque and neurofibrillary tangles, granulovacuolar degeneration is a hallmark of Alzheimer's disease. A number of tau kinases, such as c-jun N-terminal kinase (JNK), glycogen-synthase kinase-3β (GSK3β), and casein kinase 1 (CK1), have been reported to be markers of granulovacuolar degeneration. In addition, cyclin-dependent kinase 5 (CDK5), which phosphorylates tau, has been shown to be abundantly expressed in neurofibrillary tangles in the hippocampus. CDK5 has a unique staining pattern, and therefore, has the potential to be a novel marker for granulovacuolar degeneration. In this study, we investigated the ability of CDK5 to be a marker for granulovacuolar degeneration using immunohistochemical analysis. Four Alzheimer's disease cases, three myotonic dystrophy (MyD) cases, and three control cases were subjected to immunohistochemistry and immunofluorescent techniques using anti-CDK5, anti-charged multivesicular body protein 2B (CHMP2B), anti-pSmad2/3, anti-ubiquitin (Ub), anti-phospho-TDP-43 and AT8 antibodies. Some CDK5-positive granules were morphologically similar to granulovacuolar degeneration intraluminal granules, and these granules overlapped with those immunopositive for pSmad2/3, Ub and phospho-TDP-43 established granulovacuolar degeneration markers. Moreover, CDK5-positive granulovacuolar degeneration and phosphorylated tau colocalized in pyramidal neurons in Alzheimer's disease and MyD cases. The numbers of CDK5-positive granules showed an inverse relationship with the degree of mature neurofibrillary tangles in each cell, as was the case with CHMP2B-positive granulovacuolar degeneration granules and neurofibrillary tangles. The presence of tau kinases including CDK5 in granulovacuolar degeneration might implicate that granulovacuolar degeneration is structurally involved in tau modification.

Localization of CHMP2B‐immunoreactivity in the brainstem of Lewy body disease

Alpha-synuclein (αS) is one of the major constituents of Lewy bodies (LBs). Several lines of evidence suggest that the autophagy-lysosome pathway (ALP) is involved in the removal of αS. We have previously reported that granulovacuolar degeneration (GVD) in neurons involved a subunit of the endosomal sorting complexes required for transport (ESCRT). In this study, we examined the association between alpha-synucleinopathy and autophagy through immunohistochemical analysis of charged multivesicular body protein 2B (CHMP2B), a component of the ESCRT-pathway. We examined the brainstems of 17 patients with Parkinson's disease (PD), incidental Lewy body disease (ILBD), multiple system atrophy (MSA), and Alzheimer's disease (AD) immunohistochemically using antibodies against phosphorylated αS (pαS), phosphorylated tau and CHMP2B. LBs and a proportion of glial cytoplasmic inclusions (GCIs) were immunopositive for pαS and CHMP2B. Neurons containing CHMP2B-immunoreactive granules were detected in PD and ILBD, but not in MSA and AD brains. CHMP2B immunoreactivity was increased in the dorsal motor nucleus of the vagus nerve (DMNX) in PD and ILBD brains, relative to that in MSA and AD. These findings indicate that the ESCRT-pathway is implicated in the formation of αS inclusions, especially in PD and ILBD.

Impact of Less Common and “Disregarded” Neurodegenerative Pathologies on Dementia Burden in a Population-Based Cohort

Epidemiological studies investigating the pathological bases of late onset dementia focus on classical markers such as plaques and tangles. The significance of pathologies characteristically associated with rare dementia syndromes such as Pick bodies and severe neuronal loss are considered to be well defined. The significance of other pathologies, often accepted as a feature of neurodegenerative syndromes, such as Hirano bodies and gliosis is not clear. This study investigated the significance of these rarer and 'disregarded' pathologies to dementia in the population. A total of 627 individuals aged 71-103 from the Epidemiological CLInicoPathologial Studies in Europe (EClipSE) project with clinical dementia status at death determined were assessed. Pathologies assessed included Pick bodies, severe neuronal loss, gliosis, and granulovacuolar degeneration (GVD) in the cortex and/or hippocampus, along with brainstem plaques, tangles, neuronal loss, gliosis, pigmentary incontinence, and Lewy bodies. All pathologies were associated with dementia when controlling for plaques and tangles except Hirano bodies, GVD, and brainstem plaques. These included hippocampal and entorhinal gliosis; cortical, hippocampal, and entorhinal neuronal loss; along with brainstem neuronal loss, gliosis, pigmentary incontinence, Lewy bodies, and tangles. Pick bodies were present in five individuals, all with clinical dementia. These epidemiological data indicate that dementia in old age is associated with a broad range of pathological and anatomical substrates pointing to potential areas for future research, particularly the brainstem.

Lysosomal fusion dysfunction as a unifying hypothesis for Alzheimer's disease pathology.

Alzheimer's disease is characterized pathologically by extracellular senile plaques, intracellular neurofibrillary tangles, and granulovacuolar degeneration. It has been debated whether these hallmark lesions are markers or mediators of disease progression, and numerous paradigms have been proposed to explain the appearance of each lesion individually. However, the unfaltering predictability of these lesions suggests a single pathological nidus central to disease onset and progression. One of the earliest pathologies observed in Alzheimer's disease is endocytic dysfunction. Here we review the recent literature of endocytic dysfunction with particular focus on disrupted lysosomal fusion and propose it as a unifying hypothesis for the three most-studied lesions of Alzheimer's disease.

Cognitive deterioration and associated pathology induced by chronic low-level aluminum ingestion in a translational rat model provides an explanation of Alzheimer's disease, tests for susceptibility and avenues for treatment.

A translational aging rat model for chronic aluminum (Al) neurotoxicity mimics human Al exposure by ingesting Al, throughout middle age and old age, in equivalent amounts to those ingested by Americans from their food, water, and Al additives. Most rats that consumed Al in an amount equivalent to the high end of the human total dietary Al range developed severe cognitive deterioration in old age. High-stage Al accumulation occurred in the entorhinal cortical cells of origin for the perforant pathway and hippocampal CA1 cells, resulting in microtubule depletion and dendritic dieback. Analogous pathological change in humans leads to destruction of the perforant pathway and Alzheimer's disease dementia. The hippocampus is thereby isolated from neocortical input and output normally mediated by the entorhinal cortex. Additional evidence is presented that Al is involved in the formation of neurofibrillary tangles, amyloid plaques, granulovacuolar degeneration, and other pathological changes of Alzheimer's disease (AD). The shared characteristics indicate that AD is a human form of chronic Al neurotoxicity. This translational animal model provides fresh strategies for the prevention, diagnosis, and treatment of AD.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

A novel origin for granulovacuolar degeneration in aging and Alzheimer's disease: parallels to stress granules

The phosphorylated ribosomal protein S6 (pS6) is associated with the 40S ribosomal subunit in eukaryotes and is thought to have a role in RNA storage, degradation, and re-entry into translation. In this study, we found pS6 localized to granulovacuolar degeneration (GVD) within the pyramidal neurons. Immunohistochemical analysis found that nearly 20-fold more neurons contain pS6-positive granules in Alzheimer's disease (AD) hippocampus compared with age-matched controls. Further, pS6-positive granules were more common in neurons not containing neurofibrillary tangles (NFTs), were never associated with extracellular NFTs or in apoptotic neurons, and contained less RNA than neighboring pyramidal neurons not containing pS6-positive granules. In model systems, pS6 is a specific marker for stress granules, and another stress granule protein, p54/Rck, was also found to be a component of GVD in the current study. Stress granules are transient, intracellular, dense aggregations of proteins and RNAs that accumulate as a stress response, protecting cells from apoptosis and inappropriate transcriptional activity, often described as a form of ‘molecular triage.' The RNA oxidation modification 8-hydroxyguanosine (8OHG) is strikingly increased in AD, yet this study reports that those neurons with pS6 granules display reduced RNA oxidation demonstrated by lower levels of 8OHG. Since chronic oxidative stress is central to AD pathogenesis, and RNA is a specific oxidative stress target and is intimately associated with stress granule biogenesis in model systems, we suggest that GVD in human brain parallel stress granules, and may in fact be more representative of early disease pathogenesis than traditionally believed. This proposed origin for GVD as a neuroprotective response, may represent a morphologic checkpoint between cell death and reversible cellular stress that proceeds in the absence of other inclusions.

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.