Rose-Mary Na'Aman Boustany
Adjunct Professor in the Department of Pediatrics

Overview:
Our Laboratory is investigating the presence and mechanism(s) of apoptosis operative in Batten disease and other neurodegenerative diseases. So far, lipid raft structure, regulation of sphingolipid levels and their trafficking have been implicated as a theme in a number of the neuronal ceroid lipofuscinoses. We have also identified a novel Batten variant using Affymetrix GeneChip technology, and are zeroing in on the gene defect.

Batten disease is an autosomal recessive neurodegenerative disorder resulting in neuronal and photoreceptor death. We have established the presence of apoptosis in Batten disease. This was done by traditional methods such as TUNEL and PI staining, FACS analysis and electron microscopy, and by analyzing apoptosis-related genes that are dysregulated by genechip analysis.

CLN3-deficient juvenile variant: We determined that normally the CLN3 protein is made in the Golgi and travels via Rab4 and Rab11-positive recycling endosomes to lipid rafts in the plasma membrane. CLN3 harbors at its carboxyl end a number of conserved amino acids and potential glycosylation sites that are necessary for preserving antiapoptotic function and normal growth of cells. Defining these regions of the gene becomes important when one considers the facts that CLN3 is overexpressed in a number of cancers, and blocking its expression by antisense strategies results in cancer cell growth inhibition, as well as apoptotic death. These include a VYFAE motif similar to the VFFAE motif in the ß amyloid precursor mutated in some cases of familial Alzheimer disease. This motif is embedded in a structurally defined galactosylceramide lipid raft binding domain common to CLN3 protein, ß amyloid precursor, infectious prionic protein, and the V3 loop of the gp120 protein of HIV. We have shown that CLN3 protein and galactosylceramide co-localize to Golgi and in lipid rafts.
Mutant CLN3 protein in patient cells lacks this structural motif, is trapped in a fragmented Golgi, is mis-directed to Rab7-positive endosomes, and never appears in recycling endosomes or the plasma membrane. Moreover, CLN3 deficient cells have increased levels of ceramide, sphingomyelin, globoside, glucosylceramide, and galactosylceramide. Neither CLN3 nor galactosylceramide are found in lipid rafts in CLN3deficient cells. Transfection of CLN3-deficient cells with intact CLN3 cDNA restores the presence of CLN3 and galactosylceramide both to lipid rafts, and corrects ceramide levels to normal.
CLN6-deficient variant: We are in the process of defining subcellular localization of another, recently identified Batten gene called CLN6 that seems to complement CLN3-deficient cells with respect to both growth and apoptotic defects.
CLN9-deficient variant: Another novel Batten variant we call CLN9 was identified by scrutinizing genechip profiles of Batten disease cell lines. CLN9-deficient cells manifest rapid growth, increased apoptosis, a cell adhesion defect and abnormal levels of a number of complex sphingolipids including sphingomyelin, glucosylceramide, ceramide trihexoside and globoside, as well as ceramide. CLN8, the gene defective in Northern Epilepsy with Mental Retardation, complements CLN9-deficient cells with respect to growth and apoptotic defects, and partially corrects sphingomyelin levels. This suggests that CLN8 and CLN9 interact at a functional level. There is evidence that CLN3, CLN6 and CLN8 also complement each other functionally, raising the question of whether all these proteins form functional complexes.

This work will provide a handle to unravel key information pertinent to protein and lipid trafficking within the cell, and the pathobiology of these neurodegenerative diseases and may provide answers that will lead to intelligent and targeted therapies for these and other neurodegenerative diseases as well as cancer.

Current Appointments & Affiliations

Contact Information

  • 281B Med Sci Res Bldg, Durham, NC 27710
  • Box 2604 Med Ctr, Durham, NC 27710

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