We uncovered the antiapoptotic gene CLN3 by studying
the genetics, molecular and cell biology of a group
of autosomal recessively inherited diseases called
the neuronal ceroid lipofuscinoses. The two landmark
findings in my laboratory were uncovered by students.
Steven Lane, a third year medical student at the time
proved that the juvenile type of Batten disease came
about because of accelerated apoptosis. Jerry Chang,
a Duke senior, uncovered that that CLN3 was upregulated
incertain cancers. Drs. Svetlana Rylova and Wei-Xing
Guo, blocked CLN3 overexpression in these cancer cells
by an anti-sense CLN3 adenovirus that resulted in
cancer cell growth inhibition and cell death, thus
uncovering a novel target for therapeutic manipulation
in cancer. Dr. Sumeer Dhar, another post-doctoral
student in my laboratory demonstrated the effectiveness
of an available anti-apoptotic drug, flupirtine, in
blocking apoptotic death in CLN3 deficient patient
cells derived from children with the inherited neurodegenerative
disease, juvenile Batten disease. The drug also blocked
apoptotic death in number of other neurodegenerative
variants. Based on this work, we have applied for
funding to conduct a pilot clinical trial to treat
children with the late infantile form of Batten disease.
Dixie-Ann Persaud Sawin, a PhD student in the laboratory
has been examining the primary sequence of CLN3 and
has uncovered a number of highly conserved motifs
within the CLN3 protein. One of these, 291VYFAE295,
has turned out to be quite informative, It is embedded
in a highly conserved, galactocerebroside lipid raft
binding motif, common to beta amyloid, the V3 loop
of HIV gp120 protein, as well as the infectious form
of prionic protein. Dixie showed this using molecular
modeling techniques. Dixie has just completed the
study of the cellular localization of both wild type
and mutant CLN3 protein. She has found that wild type
CLN3 is found in Golgi, and recycles via rab4 and
rab11 early recycling endosomes to plasma membrane
and lipid rafts. Mutant CLN3 that lacks the GalCer
motif, however, is retained within a disrupted Golgi
and shows defective recycling. Some of the mutant
CLN3 finds its way to rab7-positive late endosomes
or lysosomes, but never makes it to the cell surface
or lipid rafts. Additionally, we found a dysregulation
of sphingolipid levels including ceramide, GalCer
and others. Defective endocytic recycling and lipd
rafts may explain the observed apoptotic defect dysregulated
sphingolipid metabolism seen in this childhood neurodegenerative
disease. Other projects in the laboratory have included
identifying the novel CLN6 and CLN9 genes, and study
of the interaction of CLN3, CLN6, CLN8, CLN9 and CLN2
genes.
