Protein Trafficking and Neurodegenerative Diseases
INITIATION DATE:
12.01.09
ARC DIRECTORS AND CO-DIRECTORS:
Lindsay Farrer, PhD; Director; Professor & Chief; Medicine/ Neurology/ Genetics & Genomics/ Epidemiology/ Biostatistics
OVERVIEW OF GOALS AND MISSION:
Recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid ß-peptide (Aß), the accumulation of which is thought central to the pathogenesis of Alzheimer disease (AD). This proposed ARC is the outgrowth of our major discovery of SORL1 as contributing to AD susceptibility (Nature Genetics 2007; 39:168-177). We observed association of several haplotypes in two distinct regions of SORL1 in seven diverse populations and showed that these variants may regulate tissue-specific expression of SORL1, which directs trafficking of APP into recycling pathways. Our work also showed that these variants may regulate tissue-specific expression of SORL1, which directs trafficking of APP into recycling pathways. When SORL1 is under-expressed, APP is sorted into Aß-generating compartments. More importantly, this body of evidence firmly establishes abnormalities and perhaps even normal variation in protein trafficking as a fundamental provocateur in AD pathogenesis. These mechanisms may involve a diverse group which regulate sorting, recycling, sequestration and metabolism of APP, other proteins which interact with APP, or intracellular structures which influence the synthesis or trafficking of APP (e.g., golgi, endoplasmic reticulum, mitochondria). This ARC will explore the role of vesicular sorting proteins and other genes involved in protein trafficking in the etiology and pathophysiology of AD and other neurodegenerative disorders. The power of this ARC lies in its ability to validate any finding using independent approaches of genetic epidemiology, cell biology, model systems and pathology. To achieve this goal, we assembled a nucleus of scientists from multiple disciplines on both campuses.
ARC Members:
MAIN ARC PROJECT(S) FOR 2009-2010:
One thrust of the ARC will consider the genetic, genomic and cell biological aspects of the relationship of trafficking proteins to AD. Another project will assess APP metabolism and endosomal transport in primary hippocampal neurons from AppSwe/PS1ΔE9 mice using immunohistochemistry and confocal microscopy. A third project will investigate the role of Klotho (a protein which declines with age in rhesus monkey, rats and mice brains and and is decreased in mouse models of AD) on trafficking and its interaction with APP and SORL1. Other projects investigating other trafficking proteins in AD and trafficking in Parkinson disease will be developed.
In one project, Dr. Farrer and Dr. Baldwin will employ mega-base sequencing to follow-up association findings from analysis of many large whole genome wide association study datasets. Mega-base sequencing is more comprehensive (virtually all SNPs evaluated) and novel SNPs (ie, those not reported in public data bases will be identified). Specifically, we will sequence 2 genes (SORL1 and ACE which are firmly established as AD risk factors) in 5 AD cases and 5 control subjects from our study of AD in Wadi Ara, an Arab community in northern Israel. Since this is a genetic isolate/inbred community, we would expect lower genetic complexity making it easier to identify the causative variants. The sequence data will be analyzed using a variety of bioinformatics tools.
Previous data from Dr. Morin’s laboratory indicate that knockdown of Vps35, a retromer subunit containing cargo recognition information, results in defective retromer transport of APP and massive accumulation of cation- independent mannose-6-phosphate receptor in HEK-293 and SH-SY5Y cells over-expressing APPSwe. Using the same lentiviral shRNA-mediated knockdown of Vps35, in a second project Dr. Morin and Dr. Ho will examine through a series of steps the effects of retromer malfunction in a double transgenic APPSwe/PS1ΔE9 mouse line, which exhibit Aβ pathology as early as 4-6 months of age. Initial studies will assess APP metabolism and endosomal transport in primary hippocampal neurons cultured from APPSwe/PS1ΔE9 mice. Cells will be plated at high-density (100,000 cells/cm2) and Aâ secretion will be measured by ELISA. Cells plated at low density (10,000/cm2) will be analyzed by confocal microscopy to assess co-localization of APP and endo-lysosomal proteins (EEA1, Rab5, Tsg101, and LAMP1). Identification of abnormal APP metabolism in cultured neurons will further support studies in APPSwe/PS1ΔE9 mice. Lentiviral vectors expressing Vps35 shRNA or a GFP marker will be injected intraventricularly in newborn transgenic APPSwe/PS1ΔE9 and control mice. Cortical amyloid deposition, cellular APP and SorL1 distribution will be assessed by immunohistological methods and confocal microscopy, respectively, at 6 months of age.
The effect of the KL C370S and F352V variants on the trafficking of both secreted and transmembrane KL will be investigated in a third project by Dr. Abraham and Dr. Chen using western blotting, subcellular fractionation, immunocyto-chemistry, and Gaussia luciferase assays to detect secretion. By gaining insight into the trafficking of KL to the PM, they hope to find ways to increase the trafficking and shedding of KL, which is associated with increased lifespan. Since the intracellular trafficking of APP is affected by SORL1, they will determine whether KL is also a substrate for SORL1 and whether the human KL polymorphisms shown to affect its trafficking and secretion are differentially recognized and processed by SORL1. Another possibility exists that SORL1 affects the secretases that process KL. To test this hypothesis Drs. Abraham and Chen will (1) Examine the effects of Klotho polymorphisms on its trafficking and shedding; (2) Examine the effects of SORL1 overexpression or knock down on the various Klotho isoforms and the α secretases trafficking and shedding; (3) Determine the mechanism by which insulin activates secretion of Klotho and APP and involvement of SORL1 in this process; (4) Test whether Klotho modifes enzymatically (as a sialidase) APP, SORL1 or the α secretases; and (5) Test novel protein trafficking-related genes identified in the GWA studies by Dr. Farrer’s group.
ARC AS A RESOURCE:
This ARC is a nidus for multi disciplinary research of neurodegenerative diseases, especially AD. Projects within the ARC will employ several high-throughput technologies including SNP-chip genotyping (Illumina platform), next-generation sequencing, and siRNA knockdown screening. Genetic and genomic data are managed and archived using state-of-the-art storage devices and capturing schema, and analyzed using BUMC’s high-performance computing system for genetics and genomics (LinGA). Members of the ARC include experts in molecular genetics, genetic epidemiology and statistical genetics, transgenic mouse models, neurology, neuropathology, neuroimaging, neurobiology, protein chemistry, proteomics, mitochondrial biology and genetics, bioinformatics, and systems biology.
Pictures, Images, Figures:
SORL1 genetic variants influence AD through cerebrovascular and neurodegenerative pathways. The A1 variant, which is near the 5’ end of SORL1 and in linkage disequilibrium (LD) with SNPs 8-10 haplotype CGC, impacts AD risk through a currently unknown pathway. Another allele at the SORL1 A locus (A2), which is in LD with SNPs 8-10 haplotype TAT, increases white matter disease (WMD) which may independently or synergistically with neurodegenerative processes lead to AD. A variant at the SORL1 B locus, which is near the 3’ end of SORL1 and in LD with SNPs 24-26, promotes AD through a neurodegenerative process causing medial temporal atrophy perhaps through the accumulation of the toxic form of amyloid β (Aβ42).
Klotho variants affect trafficking. A. Western blotting analysis of Klotho processing and secretion in COS-7 cells transiently expressing Klotho WT and variants. Note that Klotho trafficking was reduced inside (Cell lysate) and outside (Medium) for F352V variant, and increased for C370S variant. Tubulin and BSA were used as controls. Arrow indicates the 68 kDa Klotho fragment. B. Fluorescent microscopy of Klotho WT GFP and F352V GFP fusion protein. Note the strikingly different cellular distribution
CLR-derived interactome network for SORL1
- The role of Sorl1 in mediating APP localization and processing. Sorl1 associates with APP in the Golgi (1). APP and Sorl1 are endocytosed from the cell surface (2). In the absence of Sorl1, APP is delivered to a late-endosome where it can be cleaved to produce A peptides (3). At the early endosome, the retromer complex sorts Sorl1 (possibly with APP) into the endosome-to-Golgi retrieval pathway (4). Sorl1 can regulate APP localization by ‘trapping’ APP in the Golgi and/or by directing APP into the endosome-to-Golgi retrieval pathway. Image adapted from Willnow et al., 2009
(A) VPS26 knockdown (KD) results in decreased sAPP levels. (B) Retromer components VPS35 and VPS26 co-immunoprecipitate with CD8-CIMPR and CD8-SorLA (CD8-Sorl1) reporter proteins but not with CD8 or a mutant CD8-CIMPR reporter in which the Trp-Leu-Met motif is mutated to Ala-Ala-Ala.
