Jayashree Viswanathan

Down-regulation of Seladin-1 Increases BACE1 Levels and Activity through Enhanced GGA3 Depletion during Apoptosis

Seladin-1 is a neuroprotective protein selectively down-regulated in brain regions affected in Alzheimer disease (AD). Seladin-1 protects cells against β-amyloid (Aβ) peptide 42- and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the β-secretase (BACE1) function and β-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that ∼60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or Aβ secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized γ-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced β-amyloidogenic processing of APP and ultimately increased Aβ production. These adverse changes associated with decreased cell viability in seladin-1 siRNA-transfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased β-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis.

Emerging role of Alzheimer's disease-associated ubiquilin-1 in protein aggregation

Abnormal protein aggregation and intracellular or extracellular accumulation of misfolded and aggregated proteins are key events in the pathogenesis of different neurodegenerative diseases. Furthermore, endoplasmic reticulum stress and impairment of the ubiquitin-proteasome system probably contribute to neurodegeneration in these diseases. A characteristic feature of AD (Alzheimers disease) is the abnormal accumulation of Abeta (amyloid beta-peptide) in the brain. Evidence shows that the AD-associated PS (presenilin) also forms aggregates under certain conditions and that another AD-associated protein, ubiquilin-1, controls protein aggregation and deposition of aggregated proteins. Here, we review the current knowledge of ubiquilin-1 and PS in protein aggregation and related events that potentially influence neurodegeneration.

An association study between granulin gene polymorphisms and Alzheimer's disease in Finnish population†

Granulin protein plays an important role in neurite outgrowth and neuronal survival. Recently, it was shown that mutations in granulin (GRN) gene cause tau‐negative frontotemporal dementia supporting the idea that granulin is involved in neurodegeneration. Here we have investigated whether genetic variability in the GRN gene influences also the risk of developing Alzheimers disease (AD). Genotyping of six single nucleotide polymorphisms (SNPs) in the GRN gene among 512 AD patients and 649 control subjects originating from Finland did not show significant association with AD. However, stratification according to gender revealed a significant male‐specific allele, genotype and haplotype association between AD and GRN SNPs rs4792939, rs850713, and rs5848. These data suggest that genetic variability in the GRN gene may also increase the risk for developing AD in a gender‐specific manner.

Low prevalence of APP duplications in Swedish and Finnish patients with early-onset Alzheimer's disease

Familial early-onset Alzheimers disease with cerebral amyloid angiopathy (EOAD/CAA) was recently associated with duplications of the gene for the amyloid-β precursor protein (APP). In this study, we have screened for duplications of APP in patients with EOAD from Sweden and Finland. Seventy-five individuals from families with EOAD and 66 individuals with EOAD without known familial inheritance were screened by quantitative PCR. On the basis of the initial results, a portion of the samples was also investigated with quantitative multiplex PCR. No duplications of APP were identified, whereby we conclude that this is not a common cause of EOAD in the Swedish and Finnish populations, at least not in our collection of families and cases.

GRN Variant rs5848 Reduces Plasma and Brain Levels of Granulin in Alzheimer's Disease Patients

Genetic variants in the granulin (GRN) gene have been shown to increase the risk of Alzheimers disease (AD). Here, we report that the A allele of rs5848 in GRN reduces plasma granulin levels in a dose-dependent manner in a clinically-defined AD sample cohort. Similarly, the mRNA levels of granulin were decreased with respect to A allele of rs5848 in the inferior temporal cortex of neuropathologically confirmed AD patients. Our findings suggest that the A allele of rs5848 is functionally relevant by reducing the expression of granulin.

Transcriptomics and mechanistic elucidation of Alzheimer's disease risk genes in the brain and in vitro models.

In this study, we have assessed the expression and splicing status of genes involved in the pathogenesis or affecting the risk of Alzheimers disease (AD) in the postmortem inferior temporal cortex samples obtained from 60 subjects with varying degree of AD-related neurofibrillary pathology. These subjects were grouped based on neurofibrillary pathology into 3 groups: Braak stages 0-II, Braak stages III-IV, and Braak stages V-VI. We also examined the right frontal cortical biopsies obtained during life from 22 patients with idiopathic shunt-responding normal pressure hydrocephalus, a disease that displays similar pathologic alterations as seen in AD. These 22 patients were categorized according to dichotomized amyloid-β positive or negative pathology in the biopsies. We observed that the expression of FRMD4A significantly decreased, and the expression of MS4A6A significantly increased in relation to increasing AD-related neurofibrillary pathology. Moreover, the expression of 2 exons in both CLU and TREM2 significantly increased with increase in AD-related neurofibrillary pathology. However, a similar trend toward increased expression in CLU and TREM2 was observed with most of the studied exons, suggesting a global change in the expression rather than altered splicing. Correlation of gene expression with well-established AD-related factors, such as α-, β-, and γ-secretase activities, brain amyloid-β42 levels, and cerebrospinal fluid biomarkers, revealed a positive correlation between β-secretase activity and the expression of TREM2 and BIN1. In expression quantitative trait loci analysis, we did not detect significant effects of the risk alleles on gene expression or splicing. Analysis of the normal pressure hydrocephalus biopsies revealed no differences in the expression or splicing profiles of the studied genes between amyloid-β positive and negative patients. Using the protein-protein interaction-based in vitro pathway analysis tools, we found that downregulation of FRMD4A associated with increased APP-β-secretase interaction, increased amyloid-β40 secretion, and altered phosphorylation of tau. Taken together, our results suggest that the expression of FRMD4A, MS4A6A, CLU, and TREM2 is altered in relation to increasing AD-related neurofibrillary pathology, and that FRMD4A may play a role in amyloidogenic and tau-related pathways in AD. Therefore, investigation of gene expression changes in the brain and effects of the identified genes on disease-associated pathways in vitro may provide mechanistic insights on how alterations in these genes may contribute to AD pathogenesis.

Effects of Alzheimer's disease-associated risk loci on cerebrospinal fluid biomarkers and disease progression: a polygenic risk score approach.

BACKGROUND Several risk loci for Alzheimers disease (AD) have been identified during recent years in large-scale genome-wide association studies. However, little is known about the mechanisms by which these loci influence AD pathogenesis. OBJECTIVE To investigate the individual and combined risk effects of the newly identified AD loci. METHODS Association of 12 AD risk loci with AD and AD-related cerebrospinal fluid (CSF) biomarkers was assessed. Furthermore, a polygenic risk score combining the effect sizes of the top 22 risk loci in AD was calculated for each individual among the clinical and neuropathological cohorts. Effects of individual risk loci and polygenic risk scores were assessed in relation to CSF biomarker levels as well as neurofibrillary pathology and different biochemical measures related to AD pathogenesis obtained from the temporal cortex. RESULTS Polygenic risk scores associated with CSF amyloid-β42 (Aβ42) levels in the clinical cohort, and with soluble Aβ42 levels and γ-secretase activity in the neuropathological cohort. The γ-secretase effect was independent of APOE. APOE-ε4 associated with CSF Aβ42 (p < 0.001) levels. For the other risk loci, no significant associations with AD risk or CSF biomarkers were detected after multiple testing correction. CONCLUSIONS AD risk loci polygenically contribute to Aβ pathology in the CSF and temporal cortex, and this effect is potentially associated with increased γ-secretase activity.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer's disease and APdE9 transgenic mouse brain and cell-based models.

Accumulation of β-amyloid (Aβ) and phosphorylated tau in the brain are central events underlying Alzheimers disease (AD) pathogenesis. Aβ is generated from amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aβ pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aβ40 and Aβ42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aβ accumulation.

Effects of NR1H3 genetic variation on the expression of liver X receptor α and the progression of Alzheimer's disease.

Alzheimers disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42.

Ubiquilin-1 modulates γ-secretase-mediated ε-site cleavage in neuronal cells.

Ubiquilin-1 is an Alzheimers disease-associated protein, which is known to modulate amyloid precursor protein (APP) processing, amyloid-β (Aβ) secretion, and presenilin-1 (PS1) accumulation. Here, we aim to elucidate the molecular mechanisms by which full-length transcript variant 1 of ubiquilin-1 (TV1) affects APP processing and γ-secretase function in human neuroblastoma cells stably overexpressing APP (SH-SY5Y-APP751). We found that TV1 overexpression significantly increased the level of APP intracellular domain (AICD) generation. However, there was no increase in the levels of secreted Aβ40, Aβ42, or total Aβ, suggesting that ubiquilin-1 in particular enhances γ-secretase-mediated ε-site cleavage. This is supported by the finding that TV1 also significantly increased the level of intracellular domain generation of another γ-secretase substrate, leukocyte common antigen-related (LAR) phosphatase. However, in these cells, the increase in AICD levels was abolished, suggesting a preference of the γ-secretase for LAR over APP. TV2, another ubiquilin-1 variant that lacks the protein fragment encoded by exon 8, did not increase the level of AICD generation like TV1 did. The subcellular and plasma membrane localization of APP or γ-secretase complex components PS1 and nicastrin was not altered in TV1-overexpressing cells. Moreover, the effects of TV1 were not mediated by altered expression or APP binding of FE65, an adaptor protein thought to regulate AICD generation and stability. These data suggest that ubiquilin-1 modulates γ-secretase-mediated ε-site cleavage and thus may play a role in regulating γ-secretase cleavage of various substrates.