Abstract 2036 Poster Session II, Sunday, 5/2 (poster 241)

CLN3, the defective gene in the neurodegenerative disorder, juvenile Batten disease, may play a role in normal neuronal development in the central nervous system. Recently, it was determined that overexpression of CLN3 protein increased survival of NT2 neuronal precursor cells subject to apoptotic conditions. During development, cell growth is balanced with programmed cell death-apoptosis. Genes such as CLN3 may play an important role in this complex balance. With this premise, we sought to describe expression of the CLN3 gene during neuronal development. We studied 2 models. First, we evaluated weekly (over 7 weeks) expression of CLN3 in NT2 neuronal precursor cells induced with retinoic acid (10-5M) to become mature, fully differentiated post-mitotic neurons. Second, we evaluated CLN3 expression in developing brain tissue of time dated Sprague-Dawley rats during the first week of life, which is the period of rapid brain development (Preterm rat day -1, day 0, Day 4, Day 8) and at 30 days for comparison. In each model, cells and/or tissue were harvested for mRNA preparation. Using RT-PCR and 32P phosphorimaging, we quantitated the expression of CLN3 against a cyclophilin internal cellular control. We evaluated expression at the protein level using CLN3 anti-sera and Diaminobenzene (DAB) staining. In NT2 cells, formation of mature neurons was confirmed visually and by demonstration of the expression of microtubular associated protein 2B (MAP2B) and heavy neurofilament protein (NFH) expressed in the cytoskeleton. (Figure)

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In NT2 cells, expression of CLN3 was 1200 times greater in week 6 (mature neurons) than in the precursor cells. In rat brain, expression of CLN3 increased from preterm Day -1 to its greatest expression on Day 0. Levels returned to those of day -1 on day 4 and remained close to this level at days 8 and 30. The CLN3 gene was noted to be differentially expressed in the neuronal cells as they go from precursor to differentiated neurons and during rat brain development. These results were supported by protein immunohistochemistry. This differential expression of CLN3 supports a role for this gene in neuronal differentiation and maturation.