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1.  Knockdown of MLC1 in primary astrocytes causes cell vacuolation: a MLC disease cell model 
Neurobiology of disease  2011;43(1):10.1016/j.nbd.2011.03.015.
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contain other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Reexamination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.
PMCID: PMC3885813  PMID: 21440627
Astrocyte; junctions; leukodystrophy; volume
2.  EURO-WABB: an EU rare diseases registry for Wolfram syndrome, Alström syndrome and Bardet-Biedl syndrome 
BMC Pediatrics  2013;13:130.
Wolfram, Alström and Bardet-Biedl (WABB) syndromes are rare diseases with overlapping features of multiple sensory and metabolic impairments, including diabetes mellitus, which have caused diagnostic confusion. There are as yet no specific treatments available, little or no access to well characterized cohorts of patients, and limited information on the natural history of the diseases. We aim to establish a Europe-wide registry for these diseases to inform patient care and research.
EURO-WABB is an international multicenter large-scale observational study capturing longitudinal clinical and outcome data for patients with WABB diagnoses. Three hundred participants will be recruited over 3 years from different sites throughout Europe. Comprehensive clinical, genetic and patient experience data will be collated into an anonymized disease registry. Data collection will be web-based, and forms part of the project’s Virtual Research and Information Environment (VRIE). Participants who haven’t undergone genetic diagnostic testing for their condition will be able to do so via the project.
The registry data will be used to increase the understanding of the natural history of WABB diseases, to serve as an evidence base for clinical management, and to aid the identification of opportunities for intervention to stop or delay the progress of the disease. The detailed clinical characterisation will allow inclusion of patients into studies of novel treatment interventions, including targeted interventions in small scale open label studies; and enrolment into multi-national clinical trials. The registry will also support wider access to genetic testing, and encourage international collaborations for patient benefit.
PMCID: PMC3765797  PMID: 23981649
Wolfram; Alström; Bardet-Biedl; Diabetes; Patient registries; Rare diseases
3.  Erratum to 
Autophagy  2012;8(7):1163.
PMCID: PMC3429560
Lafora disease; autophagy; glycogen metabolism; laforin; malin; neurodegeneration
4.  Increased Endoplasmic Reticulum Stress and Decreased Proteasomal Function in Lafora Disease Models Lacking the Phosphatase Laforin 
PLoS ONE  2009;4(6):e5907.
Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway.
Methodology/Principal Findings
Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others.
We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.
PMCID: PMC2692001  PMID: 19529779
5.  Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility 
BMC Genomics  2008;9:12.
Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally.
This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC.
This study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.
PMCID: PMC2244606  PMID: 18190704
6.  RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods 
BMC Biochemistry  2004;5:14.
The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.
We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.
We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.
PMCID: PMC524479  PMID: 15461782

Results 1-6 (6)