Sentinel interaction mapping - a generic approach for the functional analysis of human disease gene variants using yeast.

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, 'sentinel' yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta.

BACKGROUND: Synaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid ß (Aß) peptide, and neurotoxic Aß42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aß production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer's disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins. RESULTS: Mass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca(2+)-dependent PS1 binding profiles in vitro and in vivo. We found that Aß level, and more critically, conformation of the PS1 and the Aß42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aß production at the synapse. Moreover, ß-secretase 1 (BACE1) stability, ß- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking. CONCLUSIONS: Our findings identify Syt1 as a novel Ca(2+)-sensitive PS1 modulator that could regulate synaptic Aß, opening avenues for novel and selective synapse targeting therapeutic strategies.

Oxidative stress and lipid peroxidation are upstream of amyloid pathology.

Oxidative stress is a common feature of the aging process and of many neurodegenerative disorders, including Alzheimer's disease. Understanding the direct causative relationship between oxidative stress and amyloid pathology, and determining the underlying molecular mechanisms is crucial for the development of more effective therapeutics for the disease. By employing microdialysis technique, we report local increase in the amyloid-ß42 levels and elevated amyloid-ß42/40 ratio in the interstitial fluid within 6h of direct infusion of oxidizing agents into the hippocampus of living and awake wild type mice. The increase in the amyloid-ß42/40 ratio correlated with the pathogenic conformational change of the amyloid precursor protein-cleaving enzyme, presenilin1/γ-secretase. Furthermore, we found that the product of lipid peroxidation 4-hydroxynonenal, binds to both nicastrin and BACE, differentially affecting γ- and ß-secretase activity, respectively. The present study demonstrates a direct cause-and-effect correlation between oxidative stress and altered amyloid-ß production, and provides a molecular mechanism by which naturally occurring product of lipid peroxidation may trigger generation of toxic amyloid-ß42 species.

Convergence of pathology in dementia with Lewy bodies and Alzheimer's disease: a role for the novel interaction of alpha-synuclein and presenilin 1 in disease.

A growing number of PSEN1 mutations have been associated with dementia with Lewy bodies and familial Alzheimer's disease with concomitant α-synuclein pathology. The objective of this study was to determine if PSEN1 plays a direct role in the development of α-synuclein pathology in these diseases. Using mass spectrometry, immunoelectron microscopy and fluorescence lifetime image microscopy based on Forster resonance energy transfer (FLIM-FRET) we identified α-synuclein as a novel interactor of PSEN1 in wild-type mouse brain tissue. The interaction of α-synuclein with PSEN1 was detected in post-mortem brain tissue from cognitively normal cases and was significantly increased in tissue from cases with dementia with Lewy bodies and familial Alzheimer's disease associated with known PSEN1 mutations. We confirmed an increased interaction of PSEN1 and α-synuclein in cell lines expressing well characterized familial Alzheimer's disease PSEN1 mutations, L166P and delta exon 9, and demonstrated that PSEN1 mutations associate with increased membrane association and accumulation of α-synuclein. Our data provides evidence of a molecular interaction of PSEN1 and α-synuclein that may explain the clinical and pathophysiological overlap seen in synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and some forms of Alzheimer's disease.