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1.  High Content Screening in Neurodegenerative Diseases 
The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long.
There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS.
HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility.
Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21.
Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.
PMCID: PMC3369774  PMID: 22257990
Medicine;  Issue 59;  High-throughput screening;  high-content screening;  neurodegeneration;  automated cell culturing;  Parkinson’s disease
2.  SFRS7-Mediated Splicing of Tau Exon 10 Is Directly Regulated by STOX1A in Glial Cells 
PLoS ONE  2011;6(7):e21994.
In this study, we performed a genome-wide search for effector genes bound by STOX1A, a winged helix transcription factor recently demonstrated to be involved in late onset Alzheimer's disease and affecting the amyloid processing pathway.
Methodology/Principal Findings
Our results show that out of 218 genes bound by STOX1A as identified by chromatin-immunoprecipitation followed by sequencing (ChIP-Seq), the serine/arginine-rich splicing factor 7 (SFRS7) was found to be induced, both at the mRNA and protein levels, by STOX1A after stable transfection in glial cells. The increase in SFRS7 was followed by an increase in the 4R/3R ratios of the microtubule-associated protein tau (MAPT) by differential exon 10 splicing. Secondly, STOX1A also induced expression of total tau both at the mRNA and protein levels. Upregulation of total tau expression (SFRS7-independent) and tau exon 10 splicing (SFRS7-dependent), as shown in this study to be both affected by STOX1A, is known to have implications in neurodegeneration.
Our data further supports the functional importance and central role of STOX1A in neurodegeneration.
PMCID: PMC3130792  PMID: 21755018
3.  Fine mapping of the α-T catenin gene to a quantitative trait locus on chromosome 10 in late-onset Alzheimer’s disease pedigrees 
Human molecular genetics  2003;12(23):3133-3143.
Using plasma amyloid β protein (Aβ42) levels as an intermediate, quantitative phenotype for late onset Alzheimer’s disease (LOAD), we previously obtained significant linkage at ~80 cM on chromosome 10. Linkage to the same region was obtained independently in a study of affected LOAD sib-pairs. Together, these two studies provide strong evidence for a novel LOAD locus on chromosome 10 that acts to increase Aβ42. VR22 is a large (1.7 Mb) gene located at 80 cM that encodes α-T catenin, which is a binding partner of β catenin. This makes VR22 an attractive candidate gene because β catenin interacts with presenilin 1, which has many mutations that elevate Aβ42 and cause early onset familial AD. We identified two intronic VR22 SNPs (4360 and 4783) in strong linkage disequilibrium (LD) that showed highly significant association (P = 0.0001 and 0.0006) with plasma Aβ42 in 10 extended LOAD families. This association clearly contributed to the linkage at ~80 cM because the lod scores decreased when linkage analysis was performed conditional upon the VR22 association. This association replicated in another independent set of 12 LOAD families (P = 0.04 for 4783 and P = 0.08 for 4360). Bounding of the association region using multiple SNPs showed VR22 to be the only confirmed gene within the region of association. These findings indicate that VR22 has variant(s) which influence Aβ42 and contribute to the previously reported linkage for plasma Aβ42 in LOAD families.
PMCID: PMC2836540  PMID: 14559775
4.  The R1441C mutation of LRRK2 disrupts GTP hydrolysis 
Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson’s disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate the how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.
PMCID: PMC1939973  PMID: 17442267
LRRK2; Parkinson’s disease; GTPase; kinase
5.  Analysis of IFT74 as a candidate gene for chromosome 9p-linked ALS-FTD 
BMC Neurology  2006;6:44.
A new locus for amyotrophic lateral sclerosis – frontotemporal dementia (ALS-FTD) has recently been ascribed to chromosome 9p.
We identified chromosome 9p segregating haplotypes within two families with ALS-FTD (F476 and F2) and undertook mutational screening of candidate genes within this locus.
Candidate gene sequencing at this locus revealed the presence of a disease segregating stop mutation (Q342X) in the intraflagellar transport 74 (IFT74) gene in family 476 (F476), but no mutation was detected within IFT74 in family 2 (F2). While neither family was sufficiently informative to definitively implicate or exclude IFT74 mutations as a cause of chromosome 9-linked ALS-FTD, the nature of the mutation observed within F476 (predicted to truncate the protein by 258 amino acids) led us to sequence the open reading frame of this gene in a large number of ALS and FTD cases (n = 420). An additional sequence variant (G58D) was found in a case of sporadic semantic dementia. I55L sequence variants were found in three other unrelated affected individuals, but this was also found in a single individual among 800 Human Diversity Gene Panel samples.
Confirmation of the pathogenicity of IFT74 sequence variants will require screening of other chromosome 9p-linked families.
PMCID: PMC1764752  PMID: 17166276

Results 1-5 (5)