Derivation of 5-HT neurons from human neural progenitor cells

ABSTRACTS / Experimental Neurology 198 (2006) 558 – 597

Peripheral administration of Z-lle-Glu(OtBu)-Ala-Leu-al (proteosome inhibitor; PSI) to rats has recently been shown to result in cell death in the substantia nigra (SN), locus coeruleus (LC), dorsal motor nucleus of X (DMN-X), and nucleus basalis (NB) (McNaught et al., 2004). These results indicate that systemic proteasome inhibition may closely model the pathology seen in Parkinson’s disease (PD). The current study attempts to reproduce this model in young and aged rats. Young and aged Fischer 344 (F344) and young Sprague–Dawley rats received subcutaneous injections of PSI (3 mg/kg) three times per week for 2 weeks according to previously described methods (McNaught et al., 2004). Beginning 2 weeks following the start of PSI treatment, spontaneous and amphetamine-induced locomotor activity was assessed biweekly for 6 weeks. No significant differences were observed in spontaneous or amphetamine-induced locomotor activity across all groups. Animals were sacrificed eight weeks after the initiation of PSI injections and coronal sections through the mesencephalon were immunolabeled with antisera against tyrosine hydroxylase (TH). Initial qualitative examination indicates no differences in TH immunoreactive (THir) neurons in the SN in any PSI-treated group compared to controls. Unbiased stereological analysis of THir neurons in the SN and LC, nissl histochemistry of cells in the DMN-X and p75ir neurons in the NB is currently underway. The present data suggest that systemic treatment of rats with PSI does not produce a consistent neuropathology to model Parkinson’s disease. doi:10.1016/j.expneurol.2006.02.096

Derivation of 5-HT neurons from human neural progenitor cells T. Vazin 1, L. Shen 2, J.F. Sanchez 1, C.T. Lee 1, C.J. Schoen 1, C. Spivak 1, J. Shen 2, W.J. Freed 1 1 Cellular Neurobiology Research Branch, NIDA/NIH/DHHS, Baltimore, USA 2 ScienCell Research Laboratories, San Diego, USA Recent studies have suggested that human neural progenitor cells (hNPCs) show a higher survival rate compared to postmitotic neurons in cell transplantation studies. The ability to specify the neuronal phenotype of progenitor cells is useful for transplantation studies in neurodegenerative disease paradigms. We analyzed the differentiation of hNPCs obtained from 20week human whole brain cultured as neurospheres in N2 media + bFGF/EGF. Differentiation was induced by growth factor withdrawal and addition of serum on several culture substrates. Using a marker panel of nestin, GFAP, h-III-tubulin and map2, hNPCs destined to become neurons lose nestin expression and gain h-III-tubulin expression within 1 week post-differentiation. At this time, nestin and GFAP were coexpressed in almost all h-III-tubulin negative cells. At 2 weeks post-differentiation, GFAP expression began to dominate nestin expression in h -III-tubulin negative cells, and further increased at three weeks post-differentiation as glia matured. Also at this time, h-III-tubulin positive neurons began to express the more mature neuronal marker, map2. In order to assess the phenotype

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of neurons present after 3 weeks differentiation, we screened for neurotransmitters including TH, GABA, glutamate, ChAT and 5-HT. Essentially all neurons stained positive for 5-HT, a fraction of which also co-expressed ChAT; however, GABA and glutamate were undetectable. The hNPC-derived neurons were also found to be electrically excitable and sensitive to GABA, glutamate and acetylcholine neurotransmitters. Serotonin is an important modulator of neural circuitry that controls a wide range of behavioral and physiological processes. Here we present a method for obtaining 5-HT neurons in high yield from hNPCs derived from human 20-week whole brain in vitro. A model for efficient phenotypic differentiation of serotonergic neurons could be of clinical interest in analyzing the genetics and molecular mechanisms controlling the functions of these neurons in vitro and in vivo. (This work was supported by the NIDA(IRP)/NIH/DHHS). doi:10.1016/j.expneurol.2006.02.097

´ 1 on Titer effects of lentivirus vectors expressing GABAA A audiogenic seizure behaviors D.E. Venable 1, S.A. Epps 1, S.P. Wilson 2, J.L. Fisher 2, K.C. Ross 3, J.R. Coleman 1,2 1 Department of Psychology, University of South Carolina, Columbia, USA 2 Department of Pharmacology, Physiology, and Neuroscience, School of Medicine, University of South Carolina, Columbia, USA 3 Department of Human Relations, Columbia College, Columbia, USA Altered GABA function has been implicated in the initiation of epileptic activity including a principal role by GABAA receptors. Gene transfer methods can increase specific GABAA subunit structures which can reduce epileptogenesis; this study examined effects of virus titer levels. The presumption is that subunit upregulation must effectively improve fast gating kinetics without affecting other essential supporting subunits (Fisher, J.L. and Macdonald, R.L., Neuropharm. 36, 1601– 1610 (1997)). In the first experiment, a lentivirus vector (titer: 11.6 Ag p24/ml) altering the production of GABAA a1 subunit was injected bilaterally into the central nucleus of inferior colliculus of acoustically primed Long – Evans rats exhibiting audiogenic seizure (AGS) behaviors, which include wild running and clonic convulsions. The results showed reduced incidence of clonus ( P < 0.05) and increased clonus latencies ( P < 0.05) when present, but no significant changes for wild running behavior when compared to primed non-injected controls. Next, the effects of increasing the titer (63.6 Ag p24/ ml) of the vector in the sense for a1 subunit on wild running and clonic convulsions were examined. Preliminary results show no significance for wild running or clonus parameters using the high titer vector. The sites of viral infection of target neuron populations were confirmed by immunohistochemical procedures including green fluorescent protein. These findings suggest that vector titer is an important variable in gene transfer