16.12. Alzheimer's disease-related genes and pathways

Public examination of a doctoral dissertation in the field of Molecular Medicine

Doctoral candidate: MSc (Pharm) Timo Sarajärvi

Time and venue: 16.12.2011 at 12 noon, Canthia L3, Kuopio Campus

Language of the public examination: English

Language of the dissertation: English

Alzheimer’s disease (AD) is a complex and heterogeneous disorder with an evident genetic background. So far, mutations in three genes have been implicated in the familial, autosomal dominant form of AD with an early age of onset (< 65 years). These genes are APP encoding amyloid precursor protein and PSEN1 and PSEN2 encoding presenilin-1 or presenilin-2 proteins, respectively. On the other hand, a naturally occurring polymorphism, the ε4 allele in the apolipoprotein E gene, (APOE ε4), represents the major genetic risk factor for the development of both early- (EOAD) and late-onset (LOAD) forms of AD.

Causative mutations in APP and PSEN genes explain less than 1% of all AD cases, while the vast majority of cases represent LOAD with a complex genetic inheritance. LOAD is considered to be caused by a combination of genetic, acquired, and environmental risk factors. Since the early nineties, nearly 700 individual susceptibility genes with low risk effects have been proposed as being to be associated with AD in various ethnic populations. Nonetheless, the underlying biological mechanisms of many of the risk genes in AD pathogenesis remain to be determined.

This thesis aimed at exploring the molecular mechanisms of specific genes that have been postulated to genetically and/or functionally play a role in the pathogenesis of AD. The effects of these genes on the key pathways and processes relevant for AD pathogenesis, such as generation and accumulation of beta-amyloid (Aβ), were investigated in different in vitro and in vivo models.

In this thesis, the risk effects of the common risk gene polymorphisms were assessed in the Eastern Finnish sample cohort consisting of ~1300 AD patients and controls as well as in the familial AD sample set originating from USA. In addition, the role of different factors believed to be involved in AD pathogenesis, namely seladin-1, δ-opioid receptor (δOR) and β-site APP cleaving enzyme 1 (BACE1), were examined in cultured human neuronal and non-neuronal cells as well as in the rat thalamus under conditions partially mimicking the cellular environment of the AD brain.

In study I, single nucleotide polymorphisms (SNPs) in the candidate genes were selected for genotyping on the basis of the meta-analyses retrieved from the publically accessible AlzGene database. This study revealed SNPs in the tumor necrosis factor α (TNF) and interleukin 1B (IL1B) genes, which genetically associated with AD in the Finnish case-control cohort and which also affected cerebrospinal fluid biomarker levels in AD patients.

Study II revealed that under in vitro stress conditions, reduced seladin-1/DHCR24 expression resulted in enhanced GGA3 depletion in neuronal cells. GGA3 is a well-characterized BACE1 trafficking protein, which regulates the lysosomal degradation of BACE1. GGA3 depletion subsequently led to augmented post-translational stabilization of BACE1 and increased -amyloidogenic processing of APP. This is an important mechanistic finding since the expression of seladin-1 has been consistently shown to be down-regulated in brain regions affected in AD.

Study III elucidated the genetic and functional role of δOR in AD pathogenesis. Previous data had shown that the agonist-induced activation of δOR increased the activity of β- and γ-secretases, which are responsible for Aβ generation. It was observed that the cysteine variant in the codon 27 of δOR ([1]OR-Cys27) differentially affected APP processing through altered endocytic trafficking in non-neuronal and neuronal cells as compared to the phenylalanine variant (δOR-Phe27). Furthermore, genetic assessment of the OPRD1 gene encoding for δOR in two different AD sample cohorts indicated that the heterozygosity of δOR-Phe27Cys variation may increase the risk of AD. As intervention approaches focusing on the formation and trafficking of the δOR/β- and γ -secretase complex have been suggested as novel strategies against AD, it is possible that patients with the δOR-Cys27 variant may respond to δOR antagonist treatments differentially when compared to the δOR-Phe27 homozygotes.

In study IV, a non-selective calcium channel blocker and β -secretase inhibitor, bepridil, was used to treat ischemic rats after transient middle cerebral artery occlusion (MCAO). In these MCAO rats, Aβ and calcium accumulate in the ipsilateral thalamus. Thus, MCAO represents a feasible in vivo model with which to study whether bepridil can modify Aβ and calcium pathology. Daily treatment of MCAO rats for 27 days with bepridil (50 mg/kg p.o.) starting two days after the operation decreased Aβ40 and Aβ42 and calcium levels in the ipsilateral thalamus as compared to vehicle-treated MCAO rats. Furthermore, seladin-1 mRNA levels were significantly decreased by an average of 40% in the ipsilateral thalamus of vehicle-treated, but not in the bebridil-treated MCAO rats. Significant inverse correlations between seladin-1 mRNA and calcium levels as well as with the levels of seladin-1 and insoluble Aβ42 in the ipsilateral thalamus after MCAO were observed.

Collectively, these findings suggest that in rats bepridil mitigates Aβ and calcium pathology and that seladin-1 is an indicator of neuronal survival. AD-related genes and pathways studied in this thesis may prove to be potential molecular targets and used as novel biomarkers for risk assessments, early diagnosis, and monitoring disease progression. Furthermore, the results obtained in this thesis may be applied in the development of novel intervention approaches to interrupt the progression of AD. Finally, the genetic results emphasize the importance of knowing the genetic profile of a person when introducing new potential pharmacological approaches in the therapeutics of AD.

The doctoral dissertation of Master of Science (Pharmacy) Timo Sarajärvi, entitled Alzheimer’s Disease-Related Genes and Pathways – Special Emphasis on Seladin-1, δ-Opioid Receptor and BACE1 will be examined at the Faculty of Health Sciences. The opponent in the public examination will be Associate Professor Aleister Saunders of Drexel University, USA, and the custos will be Professor Mikko Hiltunen of the University of Eastern Finland.

Photo available for download at http://www.uef.fi/vaitoskuvat

For further information, please contact: Timo Sarajärvi, tel. 040-3552518, e-mail: timo.sarajarvi@uef.fi

Publishing year: 2011

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