Poster Abstracts ISDN 2012 / Int. J. Devl Neuroscience 30 (2012) 672–692 683

possibility that the late distribution of CRc is an actively controlledprocess.

http://dx.doi.org/10.1016/j.ijdevneu.2012.10.036

ISDN2012 0216

Neuroprotection of �-Synuclein under Parkinsonian pesticideand apoptotic stimulus staurosporine treatments is diminishedby its familial Parkinson’s disease mutations

C.J. Choong, Y.H. Say ∗

Department of Biomedical Science, Universiti Tunku Abdul Rahman(UTAR) Perak Campus, MalaysiaE-mail address: sayyh@utar.edu.my (Y.H. Say).

�-Synuclein (�-Syn) plays a crucial role in the pathophysiologyof dopaminergic neurodegeneration that occurs in Parkinson’s dis-ease (PD). �-Syn has been extensively studied in many neuronalcell-based PD models but has yielded mixed results. The objec-tive of this study was to re-evaluate the dual cytotoxic/protectiveroles of �-Syn in dopaminergic human neuroblastoma SH-SY5Ycells. SH-SY5Y cells stably overexpressing wild type or familial �-Syn mutants (A30P, E46K and A53T) were subjected to acute andchronic Parkinsonian pesticides rotenone and maneb, or to apop-totic stimulus staurosporine treatments. MTT cell viability assayshowed that compared with untransfected or mock-transfectedSH-SY5Y cells, wild type �-Syn attenuated rotenone, maneb andstaurosporine-induced cell death, along with an attenuation oftoxin-induced mitochondrial membrane potential changes andReactive Oxygen Species level, whereas the mutant �-Syn con-structs exacerbated environmental toxins-induced cytotoxicityor staurosporine-induced apoptosis. After chronic treatment ofrotenone and maneb, wild type �-Syn but not the mutant vari-ants was found to rescue cells from subsequent acute hydrogenperoxide insult. Under staurosporine treatment, wild type �-Syncells have increased Bcl-2 and Bcl-XL, and decreased Bax andcleaved caspase 9 expression levels compared to the untransfected,mock-transfected and the mutants. Flow cytometric analysis withPropidium Iodide staining also revealed that the proliferative indexwas significantly reduced in cells expressing �-Syn PD mutants.Taken together, our results tend to favor the neuroprotectiveproperty of wild-type �-Syn against the toxicity of Parkinsonianenvironmental toxins rotenone and maneb, and apoptotic inducerstaurosporine. However, this neuroprotection is diminished by�-Syn familial PD mutations A30P, A53T and E46K – by causing neu-ronal cells to become more prone to oxidative and mitochondrialdamage. Thus, this ‘loss-of-function’ toxicant-mutant �-Syn inter-action could have deleterious consequences and may be involvedin the pathogenesis of PD.

http://dx.doi.org/10.1016/j.ijdevneu.2012.10.037

ISDN2012 0217

Generation of neural progenitors from human embryonic stemcell line HUES9

K. Ganapathy ∗, I. Datta

Manipal Institute of Regenerative Medicine, Manipal University, Ban-galore 560071, IndiaE-mail address: indrani.datta@manipal.edu (K. Ganapathy).

Human embryonic stem cells (huESCs)-derived neural progen-itors (NP) present an important tool for understanding humandevelopment and disease. Available methods to promote huES celldifferentiation towards neural lineages attempt to replicate, indifferent ways, the multistep process of embryonic neural devel-

opment. However, there is a need to standardize the protocol forefficient yield of neuronal progenitors with respect to (1) the pas-sage of huESCs to be considered (2) the day point of embryoidbodies to be considered for neuronal lineage and (3) the extracellu-lar matrix best suited for the generation of neural progenitors fromthe EBs. The aim of our present study was to optimize a protocolfor efficient generation of neural progenitors from human embry-onic stem cells and characterize the same. In our experience theembryoid bodies generated from the late passage huESCs were notcompact and expressed lower levels of neural progenitor markerslike nestin, Sox2, Musashi12. The EBs from early passage huESCswere well defined and expressed early neural progenitor markers.The EBs derived from both early and late passage huESCs were fur-ther characterized by real time RT-PCR at two day points for theexpression of neuronal and pluripotent markers. The day 4 EBs gen-erated from early passage huESCs expressed higher levels of nestinand Sox2. The adherence of the day 4 EBs generated from earlypassage huESCs were then examined in three matrices – gelatin,human collagen IV and matrigel. Adherence and outgrowth of neu-rites were best on human collagen IV matrix. The neural progenitorsderived on the collagen IV matrix was further expanded and char-acterized by immunofluorescence, RT-PCR and flow cytometry forearly neural stem cell markers. The neural progenitors could besub-cultured and were capable to form neurospheres in appropri-ate conditions. Flow cytometry data depicted that nearly 99% ofthe cells were positive for nestin, Musashi12 and Sox2 and neg-ative for the pluripotent marker Oct4. Therefore, the protocol forneuronal generation from human embryonic stem cells got stan-dardized with respect to the passage number, day points of EBs andefficacy of extracellular matrix. These findings suggest that our dif-ferentiation protocol has the capacity to generate efficient neuronalprogenitors from huESCs under in vitro conditions.

http://dx.doi.org/10.1016/j.ijdevneu.2012.10.038

ISDN2012 0219

Neurogenic potential of dental pulp stem cells

D. Majumdar ∗, I. Datta

Manipal Institute of Regenerative Medicine, Manipal University, Ban-galuru 560071, India

Due to its cranial neural crest origin dental pulp stem cells(DPSCs) are considered to be a better source of adult stem cellsfor cell-based therapies in neurodegenerative diseases. Recentlyfrom the perspective of translational medicine, DPSC banking hasstarted as an alternate source that is non-controversial and non-invasive. However, for DPSCs to be extensively used in cell-basedtherapies for neurodegenerative diseases, the basic need of the dayis to evaluate the neurogenic potential of these cells. To assess theneurogenic potential the present study focuses at characterizingDPSCs in neurosphere forming assay and in the presence of mid-brain cues (sonic hedgehog and fibroblast growth factor 8). Weestablished cultures of DPSCs from human adult tooth by two differ-ent isolation methods – collagenase-treatment method and explantmethod. It was observed that the collagenase method of isolatingcells showed a better yield and proliferation potential as comparedto the explant method. Further study was done to check the neu-rogenic potential of cells extracted by the collagenase treatment.The naïve DPSCs spontaneously expressed both early and late neu-ronal markers and on being treated with neural stem cell media innon-adherent conditions, it was capable to form neurosphere likestructures. Quantitative RT-PCR results depicted that the early neu-ronal marker nestin was upregulated and mature neuronal marker� tubulin III was downregulated in the neurosphere like structure.The neurosphere like structures were also positive for pluripotent