SEPT8 modulates ß-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.

Dysfunction and loss of synapses are early pathogenic events in Alzheimer's disease. A central step in the generation of toxic amyloid-ß (Aß) peptides is the cleavage of amyloid precursor protein (APP) by ß-site APP-cleaving enzyme (BACE1). Here, we have elucidated whether downregulation of septin (SEPT) protein family members, which are implicated in synaptic plasticity and vesicular trafficking, affects APP processing and Aß generation. SEPT8 was found to reduce soluble APPß and Aß levels in neuronal cells through a post-translational mechanism leading to decreased levels of BACE1 protein. In the human temporal cortex, we identified alterations in the expression of specific SEPT8 transcript variants in a manner that correlated with Alzheimer's-disease-related neurofibrillary pathology. These changes were associated with altered ß-secretase activity. We also discovered that the overexpression of a specific Alzheimer's-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aß peptides in neuronal cells. These changes were related to an increased half-life of BACE1 and the localization of BACE1 in recycling endosomes. These data suggest that SEPT8 modulates ß-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1.

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

Accumulation of ß-amyloid (Aß) and phosphorylated tau in the brain are central events underlying Alzheimer's disease (AD) pathogenesis. Aß is generated from amyloid precursor protein (APP) by ß-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 Aß 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 Aß40 and Aß42. 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 Aß accumulation.

Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation.

The Alzheimer's disease (AD)-associated ubiquilin-1 regulates proteasomal degradation of proteins, including presenilin (PS). PS-dependent γ-secretase generates ß-amyloid (Aß) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin-1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ-secretase complex components and subsequent γ-secretase function in human embryonic kidney 293, human neuroblastoma SH-SY5Y and mouse primary cortical cells. Full-length ubiquilin-1 TV1 and TV3 that lacks the proteasome-interaction domain increased full-length PS1 levels as well as induced accumulation of high-molecular-weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1- and TV3-expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitin-proteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aß levels, particularly in cells overexpressing TV3. These effects were not related to altered γ-secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin-1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

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

Ubiquilin-1 is an Alzheimer's disease-associated protein, which is known to modulate amyloid precursor protein (APP) processing, amyloid-ß (Aß) 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 Aß40, Aß42, or total Aß, 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.

3rd Annual FinnProt Meeting: from cells to systems.

The Finnish Proteomics Society, FinnProt ( www.finnprot.org ), was founded in November 2004 as the Proteomics Division of the Societas biochemica, biophysica et microbiologica Fenniae ( www.biobio.org ). The mission of FinnProt is to make proteomics research readily available for the large scientific community in Finland, promote research and education in proteomics and protein chemistry, and act as the official Finnish collaborative body to international proteomics organizations such as the European Proteomics Association.

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 Alzheimer's 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-ß 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-, ß-, and γ-secretase activities, brain amyloid-ß42 levels, and cerebrospinal fluid biomarkers, revealed a positive correlation between ß-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-ß 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-ß-secretase interaction, increased amyloid-ß40 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 Alzheimer's 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-ß42 (Aß42) levels in the clinical cohort, and with soluble Aß42 levels and γ-secretase activity in the neuropathological cohort. The γ-secretase effect was independent of APOE. APOE-ε4 associated with CSF Aß42 (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 Aß pathology in the CSF and temporal cortex, and this effect is potentially associated with increased γ-secretase activity.

High-fat diet increases tau expression in the brain of T2DM and AD mice independently of peripheral metabolic status.

Alzheimer's disease and type 2 diabetes mellitus are risk factors for each other. To investigate the effects of both genetic and high-fat-induced diabetic phenotype on the expression and exon 10 splicing of tau, we used the Alzheimer's disease mouse model (APdE9) cross-bred with the type 2 diabetes mouse model over-expressing insulin-like growth factor 2 in the pancreas. High-fat diet, regardless of the genotype, significantly induced the expression of four repeat tau mRNA and protein in the temporal cortex of female mice. The mRNA levels of three repeat tau were also significantly increased by high-fat diet in the temporal cortex, although three repeat tau expression was considerably lower as compared to four repeat tau. Moreover, high-fat diet significantly increased the mRNA ratio of four repeat tau vs. three repeat tau in the temporal cortex of these mice. All of these effects were independent of the peripheral hyperglycemia, hyperinsulinemia and insulin resistance. Increased four repeat tau and three repeat tau levels significantly associated with impaired memory and reduced rearing in the female mice. High-fat diet did not affect neuroinflammation, Akt/GSK3ß signaling pathway or the expression of tau exon 10 splicing enhancers in the temporal cortex. Our study suggests that the high-fat diet independently of type 2 diabetes or Alzheimer's disease background induces the expression and exon 10 inclusion of tau in the brain of female mice.

Targeting ApoE4/ApoE receptor LRP1 in Alzheimer's disease.

INTRODUCTION: Progressive neuronal loss is a key feature in Alzheimer's disease (AD), which is the most common neurodegenerative disorder in the aging population. Currently, there are no therapeutic means to intervene neuronal damage in AD and therefore innovative approaches to discover novel strategies for the treatment of AD are needed. Based on the prevailing amyloid cascade hypothesis, it is conceivable that lowering the ß-amyloid (Aß) levels is sufficient to slow down the disease process, if started early enough. AREAS COVERED: Here, we review genetic and biological functions related to apolipoprotein E (ApoE) and low-density lipoprotein receptor-related protein 1 receptor (LRP1)-mediated clearance of Aß. Furthermore, we discuss the AD-related therapeutic potential of targeting to ApoE receptor LRP1 at the blood-brain barrier (BBB) and in the periphery. EXPERT OPINION: Due to the recent setbacks in the clinical trials targeting AD, it is instrumental to seek alternative therapeutic approaches, which aim to reduce the accumulation of Aß in the brain tissue. As the ApoE/LRP1-mediated clearance of Aß across the BBB is the key event in the regulation of Aß transcytosis from brain to periphery, direct targeting of this protein entity at the BBB holds a great potential in the treatment of AD.

Elucidation of the BACE1 regulating factor GGA3 in Alzheimer's disease.

Golgi-localized γ-ear-containing ADP-ribosylation factor-binding protein (GGA3) is a central regulator of trafficking and degradation of BACE1 (ß-site AßPP-cleaving enzyme), the rate-limiting enzyme in the production of amyloid-ß (Aß) in Alzheimer's disease (AD). Here, we assessed the potential role of GGA3 in AD pathogenesis using independent neuropathological, case-control, and family-based human sample cohorts. Increased BACE1 levels coincided with decreased GGA3 levels and with elevated phosphorylation status of eIF2α-Ser51 in the temporal cortex of AD patients as compared to age-matched controls. Severity of the disease did not alter mRNA or protein levels of GGA3 in the inferior temporal cortex of AD patients, while a positive correlation between GGA3 and the levels of total, but not phosphorylated, tau was observed. Genetically, we did not observe consistent evidence for association between AD risk and common GGA3 polymorphisms across a number of independent sample cohorts. However, a nominally significant association was observed with rs2242230 (p < 0.05) among the Finnish case-control cohort. Accordingly, mRNA and protein levels of GGA3 in the inferior temporal cortex of AD patients did not significantly correlate with rs2242230 genotype status. While the present study indicates that GGA3 is involved in the cellular processes relevant for AD pathogenesis, the genetic data do not support the idea that common GGA3 polymorphisms would contribute to AD risk.