The Retromer Pathway Links Obesity and Diabetes to Alzheimer's Disease

Featured Research Sessions F2-01: Protein Sorting and Trafficking in Alzheimer’s Disease: Genes, Pathways and Pathogenesis phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. This APP S655E phosphomimetic mutant also displayed decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. It follows that VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life. Conclusions: These results permit concluding that APP phosphorylation on S655 regulates its fate in terms of retromer-mediated sorting to the TGN or lysosomal targeting. Furthermore, the data are consistent with known interactions involving the retromer, SorLA and APP. These findings add to our understanding of APP targeting and the molecular basis of sporadic AD pathogenesis; representing putative new targets for AD diagnostic and therapeutic approaches.

F2-01-04

A NETWORK OF PROTEIN KINASES AND SORTILINS CONVERGES ON VPS35 TO CONTROL A GENERATION

Sam Gandy, Mount Sinai, New York, N.Y., United States. Background: APP sorting and beta-amyloid generation are dynamically regulated by the integration of neurotransmitter and hormone signals that modulate the phosphorylation status of a host of potential effector substrate proteins. SorCS1 and SorL1 belong to the sortilin family of proteins and are two such potential effector phosphoproteins. Both of these sortilins are genetically associated with Alzheimer’s disease (AD), and SORCS1 locus is associated with insulin resistance and type 2 diabetes mellitus (T2DM) in addition to its association with AD. SORCS1 linkage to AD and T2DM show sexual dimorphism. Methods: In some experiments, HEK293 cells were transfected with various combinations of plasmids for APP, SORCS1 isoform enriched in the brain, SorL1, Rho kinase 2 (ROCK2), or appropriate control plasmids. Intracellular and extracellular APP metabolites were determined and quantified in cellular lysates, conditioned media, and brain homogenates. Protein-protein complexes were determined using co-immunoprecipitation/immunoblotting (co-IP/IB) from either transfected cells or native tissue. Results: SorL1 was identified as a phosphoprotein that undergoes PKC-sensitive ectodomain shedding. ROCK2 and SorCS1 were identified as novel regulators of beta-amyloid metabolism, acting via mechanisms involving SorL1. Secreted beta-amyloid levels were reduced by overexpression of SorCS1; conversely, secreted A beta-amyloid levels were increased by knockdown of ROCK2 using a specific shRNA. We hypothesized that the changes in beta-amyloid generation might be attributable to identifiable protein: protein interactions. Using coIP/IB, a number of pairwise and tripartite protein complexes were recovered from transfected and native tissue, including [SorCS1:SorL1:APP] complexes and [SorL1:ROCK2] complexes. In support of the in vivo relevance of these interactions, levels of A€ı ¢ were observed to be elevated in the brains of female (but not male) Sorcs1-deficient mice, paralleling the sexual dimorphism observed in the genetic linkage to both AD and T2DM. Conclusions: SorL1 is phosphoprotein that binds APP, SorCS1, ROCK2, and Vps35, creating a protein interaction network converging on the Vps35 component of the retromer. Notably among these SorL1-binding proteins is SorCS1, which is linked to insulin resistance and T2DM. Because of the linkage of SorCS1 to both AD and T2DM, we hypothesize that SorCS1 will provide an important point of entry for dissecting the molecular basis for the increased risk of AD associated with T2DM. A detailed elucidation of the mechanisms linking these modulators of A€ı ¢ generation to the integrity of the SorL1:Vps35 complex may lead to novel insights about the underpinnings and/or therapy of AD. F2-01-05

GENES FOUND BY GWAS IN ALZHEIMER’S DISEASE: COMPARING VPS10-CONTAINING RECEPTORS WITH BIN1 AND CALM1

Peter St. George Hyslop, University of Toronto, Toronto, ON, Canada.

S283

Background: Recent Genome-Wide Association Studies have identified two genes (PICALM and BIN1) involved in physiological vesicular trafficking as potentially harbouring genetic variants associated with risk for late onset sporadic Alzheimer’s disease. Several other genes involved in vesicular trafficking (SORL1 SORCS1, SORCS2) have been associated with risk for AD, and have apparently caused this risk by altering APP processing, and increasing the production of Abeta. We therefore hypothesised that that these other genes might also alter APP trafficking, particularly trafficking related to synaptic activity and synaptic vesicle recycling in neurons. Methods: PICALM and BIN1 cDNAs, and PICALM and BIN1 shRNAs were cloned into Lentiviral expression vectors that provided either constitutive expression or inducible expression under tetracycline control. Stable cell lines were then generated using a variety of cell types (e.g. HEK293, HeLa, SHSY5Y). We then measured Abeta and APPs_alpha, APPs_beta levels in the conditioned media and in cell lysates, and we investigated the levels of mature and immature APP in the cell lysates. Results: In cell lines expressing PICALM shRNA molecules, there was a small (20%), but consistent reduction in Abeta secretion. The effects of PICALM overexpression are currently being investigated. In cell lines expressing wild-type APP and BIN1 shRNAs, there was no consistent alteration in Abeta secretion or on APP processing. Similarly, in cell lines over-expressing wild-type APP and BIN1 cDNAs, there was also no consistent alteration in Abeta secretion or on APP processing. However, in cell lines co-expressing APP_swedish, although there were no changes in Abeta secretion, there were subtle changes in APPs_alpha levels after either BIN1 suppression or BIN1 overexpression, suggesting that while BIN1 had no effect on APP entering into the late endosome–lysosome pathway for BACE and gamma-secretase cleavage, it did affect APP_Swedish processing on the cell surface. Conclusions: In strong contrast to the effects of the overexpression/knockdown of SORL1, SORCS1, SORC2, and other genes involved in the retromer pathway, modulation of PICALM and BIN1 expression have only modest effects on APP processing. These results, which will need to be confirmed by further studies, suggest several possible conclusions. First, while the SNPs associated with AD on GWAS fall within/close to the BIN1 and PICALM genes, it has not yet been proven that BIN1 and PICALM are in fact the functional elements impacted by these disease-associated SNPs. It is currently still formally possible that the SNPs in BIN1 and PICALM genes might actually influence the expression of other genes nearby. Second, it is possible that the disease-causing SNPs do alter BIN1 and PICALM function, but do so by causing a gain-of-toxic function which is not predictable via simple up- or down-regulation of BIN1 and PICALM expression. Finally, it is possible that the disease-causing SNPs do effect BIN1 and PICALM function, but this function does not modulate APP processing and Abeta production, but might affect some other downstream aspect of Abeta toxicity. F2-01-06

THE RETROMER PATHWAY LINKS OBESITY AND DIABETES TO ALZHEIMER’S DISEASE

Scott Small, Columbia University, New York, N.Y., United States. Background: VPS35 and VPS26 is deficient in late-onset Alzheimer’s disease (AD), and this is linked to deficiencies in VPS10-receptors, sorL1 and sorCS1. These molecules are all part of the retromer sorting & trafficking pathway. Studies have established how deficiencies in this sorting & trafficking pathway contribute to the pathogenesis of AD. Although genetic defects can underlie retromer dysfunction in rare cases, we reasoned that there must be additional ‘environmental’ factors that contribute to the observed deficiencies in AD. Based on previous studies, we hypothesized that defects in obesity and type II diabetes might cause deficiencies in the retromer pathway. Methods: We measured VPS35, VPS26, and sorL1 in the hippocampus and cerebellum of OB/OB mice, a mouse model of obesity and type II diabetes. In secondary analysis we also used ELISA to measure endogenous abeta and western blot to measure tau phosphorylation. Results: We find that VPS35, VPS26 and sorL1 are deficient in OB/OB mice, selectively in the hippocampus but not in the

S284

Featured Research Sessions F2-02: The Interaction of Alzheimer’s Disease and Vascular Disease on Cognition

cerebellum. Mild accumulation of both abeta40 and abeta42 was observed, as was significant tau hyperphosphorylation. Preliminary studies suggest that abnormal serological levels of branched-chain amino acids mediate this effect. Conclusions: Obesity and type II diabetes are emerging as an epidemic, and have been epidemiologically linked to AD. Our current observations suggest that the induced deficiencies in the retromer pathway can, in part, mediate this link. We are currently investigating cellular and molecular mechanisms by which serological abnormalities observed in obesity and type II diabetes cause deficiencies in the retromer sorting & trafficking pathway. MONDAY, JULY 18, 2011 FEATURED RESEARCH SESSIONS F2-02 THE INTERACTION OF ALZHEIMER’S DISEASE AND VASCULAR DISEASE ON COGNITION F2-02-01

RELATION OF ALZHEIMER’S DISEASE AND CVD PATHOLOGY ON THE EXPRESSION OF COGNITIVE IMPAIRMENT

David Bennett, Rush University Medical Center, Chicago, Ill., United States. Background: To the examine the relation of measures of Alzheimer’s Disease (AD) pathology and cerebrovascular disease to cognitive impairment in older persons with and without dementia. Methods: More than 2,600 older persons without dementia who agreed to organ donation as part of the Religious Orders Study and the Rush Memory and Aging Project underwent annual detailed clinical evaluation. More than 850 have died and undergone brain autopsy at which time measures of AD pathology (diffuse and neuritic plaques, and neurofibrillary tangles) was quantified, amyloid angiopathy was assessed, as was the number, volume and location of macroscopic infarctions, the number of microscopic infarctions, and arteriolar sclerosis. Regression analyses were used to examine cognition and dementia as a function of AD and CVD pathology. Results: Nearly every brain had some evidence of AD pathology. Macroscopic infarcts were present in about a third and microscopic in about a third, with half also having macroscopic infarcts. Infarcts were not related to AD pathology. AD pathology, macroscopic infarctions and microscopic infarcts all had relatively independent effects on level of cognition. Further there was no evidence of effect modification. Similar findings were seen in models with clinically diagnosed dementia. Conclusions: AD and CVD both contribute to cognitive impairment. The prevention of CVD would reduce the occurrence of dementia.

Conclusions: The finding that vascular risk factors increase the risk of AD may have implications for prevention, and my shed light on the pathogenesis of this disease. F2-02-03

Bruce R. Reed, UC Davis Alzheimer’s Center, Martinez, Calif., United States. Background: Advancing aging is associated with an increased prevalence in vascular risk factors, symptomatic vascular disease and Alzheimer’s disease (AD). It is likely, therefore, that individuals diagnosed with AD will have concomitant vascular disease, including AD patients involved in clinical trials. Methods: MRI is a method sensitive to the presence of vascular brain injury that can be used as an in vivo measure to understand the interaction between cerebrovascular disease (CVD) and AD. Our laboratory has used advanced image processing methods to examine the impact of vascular risk factors and vascular brain injury as measured by the presence of MRI infarction and white matter hyperintensities (WMH). Results: Advancing age is associated with increased prevalence of asymptomatic cerebral infarction and extent of WMH. Each of these measures is associated with increased risk for stroke, dementia and mortality among community dwelling healthy older individuals. In addition, vascular risk factors such as hypertension, diabetes and cigarette smoking are associated with increased rate of WMH accretion, brain atrophy and decline in measures of executive function. fMRI studies of cognitively normal older individuals with high WMH volumes show reductions in prefrontal activation to a variety of cognitive tasks suggesting that vascular brain injury may alter cognition through impairment of frontal systems. In addition, executive function interacts with medial temporal integrity to predict episodic memory performance. These results were further refined through analysis of diffusion tensor imaging in relationship to cognitive performance. Finally, recent work finds that WMH, brain atrophy and the strength of resting state fMRI connectivity independently predict memory performance. WMH are also increased in extent and distribution among individuals with mild cognitive impairment (MCI) and are associated with an increased likelihood of conversion to dementia. Finally, in the ADNI study, we show that WMH account for significant decline in MMSE and ADAS-Cog when accounting for measures of global and regional atrophy. Preliminary data utilizing the combination of MRI and PiB imaging reveals that CVD has an independent effect on cognition. Conclusions: In conclusion, increasing evidence suggests that MRI measures of CVD independently affect cognition with advancing age and in the setting of MCI and clinically diagnosed AD. F2-02-04

F2-02-02

VASCULAR DISEASE RISK FACTORS AND ALZHEIMER’S DISEASE

MRI MARKERS OF VASCULAR DISEASE AND THEIR RELATIONSHIP TO COGNITION

IMAGING AMYLOID AND CEREBROVASCULAR DISEASE IN AGING

Ingmar Skoog, University of Gothenburg, Gothenburg, Sweden.

Natalie Marchant, Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, Calf., United States.

Background: To examine the evidence that vascular risk factors are important in the etiology of Alzheimer’s disease (AD). Methods: Literature review and results from the population studies in Gothenburg, Sweden, including the prospective population study of Women where 1468 women have been followed from 1968 to 2010, the H70-study comprising two samples of 70-year-olds born 1901-02 (followed until age 105) and 1930 (followed until age 79), H85 comprising two samples of 85-year-olds born 1901-02 (followed until age 105) and 1923-24, and 95+ comprising 950 individuals aged 95- years and over. Results: Vascular risk factors (such as hypertension, overweight, hyperhomocysteinemia, diabetes mellitus, and stress) seem to increase risk for AD in late life. On the other hand, factors protective of vascular disease (such as moderate wine consumption, good lung function and exercise) seems to be protective for AD. Some of these relationships are rather complex, e.g. both high and low blood pressure seems to increase risk for AD. Furthermore, secular trends in the frequency of vascular risk factors may influence the frequency of AD in the future.

Background: Cerebrovascular disease may exert effects on the brain directly via stroke and ischemia. Cerebrovascular risk factors are also known to increase the risk of clinical AD. While both risk factors and stroke are measureable during life, until recently the assessment of AD pathology in vivo was not possible. This has changed with the advent of amyloid imaging. This talk will report on studies conducted to understand the relationships between cerebrovascular disease, cerebrovascular risk, Abeta pathology, and cognition. Methods: We have recruited several cohorts of individuals with a wide range of cerebrovascular pathology including those with extensive white matter hyperintensities (WMH) and infarction. Subjects are categorized for risk using the Framingham Coronary Risk Profile (FCRP) score. Subjects are also categorized as having evidence of cerebrovascular disease (CVD+) by virtue of either extensive WMH or infarction measured with MRI. Abeta pathology was measured with [11C]PIB, quantified as both a continuous and categorical measure. Results: In one study of 43 individuals comprised of subjects ranging from cognitively normal to demented,