Teemu Natunen

Genetic loci associated with Alzheimer's disease and cerebrospinal fluid biomarkers in a Finnish case-control cohort.

Objectives To understand the relation between risk genes for Alzheimer’s disease (AD) and their influence on biomarkers for AD, we examined the association of AD in the Finnish cohort with single nucleotide polymorphisms (SNPs) from top AlzGene loci, genome-wide association studies (GWAS), and candidate gene studies; and tested the correlation between these SNPs and AD markers Aβ1–42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF). Methods We tested 25 SNPs for genetic association with clinical AD in our cohort comprised of 890 AD patients and 701-age matched healthy controls using logistic regression. For the correlational study with biomarkers, we tested 36 SNPs in a subset of 222 AD patients with available CSF using mixed models. Statistical analyses were adjusted for age, gender and APOE status. False discovery rate for multiple testing was applied. All participants were from academic hospital and research institutions in Finland. Results APOE-ε4, CLU rs11136000, and MS4A4A rs2304933 correlated with significantly decreased Aβ1–42 (corrected p<0.05). At an uncorrected p<0.05, PPP3R1 rs1868402 and MAPT rs2435211 were related with increased t-tau; while SORL1 rs73595277 and MAPT rs16940758, with increased p-tau. Only TOMM40 rs2075650 showed association with clinical AD after adjusting for APOE-ε4 (p = 0.007), but not after multiple test correction (p>0.05). Conclusions We provide evidence that APOE-ε4, CLU and MS4A4A, which have been identified in GWAS to be associated with AD, also significantly reduced CSF Aβ1–42 in AD. None of the other AlzGene and GWAS loci showed significant effects on CSF tau. The effects of other SNPs on CSF biomarkers and clinical AD diagnosis did not reach statistical significance. Our findings suggest that APOE-ε4, CLU and MS4A4A influence both AD risk and CSF Aβ1–42.

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.

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 (β-site AβPP-cleaving enzyme), the rate-limiting enzyme in the production of amyloid-β (Aβ) in Alzheimers 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.

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.

Genetic analysis of genes involved in amyloid-β degradation and clearance in Alzheimer's disease.

Accumulation of amyloid β-peptide (Aβ) in the brain of Alzheimers disease (AD) patients has been postulated to reflect defects in Aβ degradation or clearance. Here, we selected 12 genes (MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9) involved in Aβ catabolism on the basis of PubMed-based literature search and elucidated their genetic role in AD among Finnish case-control cohort consisting of total ∼1,300 AD patients and control subjects. Thirty one single nucleotide polymorphisms (SNPs) were selected for genotyping. In a smaller subset of AD patients, cerebrospinal fluid (CSF) levels of Aβ42 (n = 124), total-tau (n = 59), and phospho-tau (n = 54) analyses were performed with respect to SNPs. Moreover, age of onset analyses with respect to the studied SNPs were conducted among the AD patient cohort (n = 642). Association analysis of the liver X receptor α (NR1H3) gene SNPs showed a protective effect for C allele carriers of rs7120118 (OR = 0.70, 95% CI 0.53-0.93), while the total-tau and phospho-tau levels in CSF were decreased in AD patients carrying the C allele. Also, a decrease in the age of onset was observed in AD patients carrying the A allele of rs723744 and the C allele of rs3794884 in transthyretin (TTR) gene. However, after adjusting the p-values for multiple comparisons, these results were not statistically significant, suggesting that genetic variations in MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9 genes do not play major role among the Finnish AD patient cohort.

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

ABSTRACT Dysfunction and loss of synapses are early pathogenic events in Alzheimers disease. A central step in the generation of toxic amyloid-β (Aβ) peptides is the cleavage of amyloid precursor protein (APP) by β-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 Aβ generation. SEPT8 was found to reduce soluble APPβ and Aβ 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 Alzheimers-disease-related neurofibrillary pathology. These changes were associated with altered β-secretase activity. We also discovered that the overexpression of a specific Alzheimers-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aβ 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 β-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1. Summary: The transcript variant ratio of SEPT8 is altered according to the Alzheimers-disease-related neurofibrillary pathology. At the cellular level, SEPT8 modulates β-amyloidogenic processing of APP by affecting BACE1.