The long term goals of the proposed research are to elucidate the mechanisms governing neuronal proliferation and determination of brain size. Mutations in genes causing microcephaly encode proteins that localize to the centrosome and pericentriolar matrix, suggesting that this is the seat of an important common pathway that regulates neuronal progenitor mitosis. However, the precise mechanism by which these proteins affect proliferation is unclear. Our lab has recently identified a novel mutation in a centrosomal protein in a consanguineous Palestinian family affected by severe microcephaly, lissencephaly and intellectual disability. The major goal of this proposal is to study the role of this protein i regulating neuronal progenitor mitosis and cell cycle progression in the developing mammalian cerebral cortex.
Specific Aim 1 will characterize the novel mutation using RT-PCR and Western blot analysis to assay for abnormal mRNA transcripts and protein products in patient lymphoblasts. Preliminary mini- gene splicing assays show the mutation generates an mRNA transcript that skips an in-frame exon, producing a smaller, less stable protein product.
Specific Aim 2 will study the cell biological role of the protein and test the hypotheses that its loss of function will cause mitotic arrest, either by disrupting spindle architecture or ciliary resorption and that this may represent a common effector pathway for other microcephaly genes, such as NDE1, which exhibits a strikingly similar phenotype. This will accomplished by RNAi knockdown in cell culture to assay for prolonged cell cycle length, abnormal mitotic spindle organization, changes in ciliary length and functional and biochemical interaction with Nde1. Finally, Specific Aim 3 will study the effect of loss of function in vivo using a knockout mouse model. Mutant mice will be analyzed for changes in brain histology using immunohistochemistry for progenitor, neuronal subtype and cortical layer specific markers, and apoptotic markers, at various time points during embryonic and early postnatal development;and for premature progenitor cell cycle exit using BrdU pulse experiments, coupled with Ki67 staining. Together, these experiments will provide further insights into the mechanisms regulating neuronal progenitor proliferation during normal cortical development and how their deregulation causes developmental neurological disorders.

Public Health Relevance

The proposed research represents an important series of experiments to advance our understanding of normal brain development. As deregulation of proliferation, migration and cell fate specification cause multiple cortical malformations and impair cognitive function in patients, the proposed studies will further elucidate the mechanisms underlying developmental neurological disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31NS083111-02
Application #
8658299
Study Section
NST-2 Subcommittee (NST)
Program Officer
Riddle, Robert D
Project Start
2013-05-01
Project End
2015-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
2
Fiscal Year
2014
Total Cost
$34,338
Indirect Cost
Name
Harvard University
Department
None
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Hu, Wen F; Chahrour, Maria H; Walsh, Christopher A (2014) The diverse genetic landscape of neurodevelopmental disorders. Annu Rev Genomics Hum Genet 15:195-213