The Effect Of Alpha-synuclein And Its PD-associated Mutations On Dopaminergic Neuron Differentiation In Human Embryonic Stem Cell

Parkinson's disease (PD) is the most common progressive neurodegenerative movement disorder in central nervous system(CNS).α-synuclein is the first gene identified in patients with PD. The biological function ofα-synuclein is not well understood though numerous studies have demonstrated that it may play important roles in regulation of synaptic plasticity, recycling of synaptic vesicles, storage, distribution of neurotransmitter and differentiation of neurons. Recent studies onα-synuclein are mainly focused on the aggregation and toxicity of wild type and its pathogenic mutations in PD(A30P, E46K, A53T), the contribution of pathogenic mutations to oxidative damage, mitochondrial dysfunction, the dysfunction of ubiquitin-proteasome system and abnormal autophagy. The effect ofα-synuclein and its PD-associated mutations on the development of neural system, especially on the differentiation of dopaminergic neuron are still lack of thorough study for lacking of an ideal model. Human embryonic stem cells(hESCs) have the ability to undergo both self-renewal and differentiation. Right now the differentiation of hESCs into dopaminergic neurons has become a mature technique, which can be used as a window to reflect the process of dopaminergic neuron differentiation in vivo. This technique, consequently, can also be used to reveal certain impacts of the wild typeα-synuclein and its PD pathogenic mutations (A30P, E46K, A53T) on dopaminergic neuron differentiation.Aims:Obtain hESCs cell lines stably expressing wild type a-synuclein and its PD pathogenic mutations (A30P,A53T).Methods:The lentivirus of wild type a-synuclein and its PD pathogenic mutations (A30P,A53T) were derivated from co-transfection of WT,A30P, A53T a-synuclein-FUIGW-GFP with helper plasmids into 293FT cells.48 and 72 hours after transfection, virus-containing supernatant was collected and concentrated by ultracentrifue, and then use the concentrated lentivirus to infect hESCs (H9). After infection hESCs with GFP fluorescence were picked up and expanded. For the disparities of each clump we identify the progenies of the same clump as a cell lineage. At each passage, hESCs cell lineages stably expressing a-synuclein and its pathogenic mutations in PD (A30P, A53T) were purified according to GFP fluorescence. The stem cell markers were detected and embryoid bodies (EB) formation in vitro as well as the teratoma formation in vivo were also did to demonstrate the biological features of human embryonic stem cell.Results:The GFP fluorescence appeared at approximate 7days after infection. After that hESCs with GFP fluorescence were picked up and expanded. For the disparities of each clump we identify the progenies of the same clump as a cell lineage. At each passage, hESCs cell lineages stably expressing a-synuclein and its pathogenic mutations in PD (A30P, A53T) were purified according to GFP fluorescence. HESC stably expressing a-synuclein and its PD-associated mutations (A30P, A53T) all express the stem cell markers and besides that, they can form EB in vitro and teratoma in vivo.Conclusion:(1) These H9 sub-lineages stably expressing a-synuclein and PD-associated mutations(A30P, A53T) retained the biological features of hESCs. They have the capacity to differentiate both in vitro and in vivo. (2) Wild typeα-synuclein's and pathogenic mutations in PD (A30P, A53T) showed a diffuse and punctate distribution in cytoplasm and nucleus at early passage. However with the continue culture wild type and A53Tα-synuclein were distributed on the membrane of a vesicle-like structure in cytoplasm.Aims:Compare the differences betweenα-synuclein and its PD associated mutations (A30P, A53T) in the differentiation of dopaminergic neurons.Methods:To obtain H9 sub-lineages stably expressingα-synuclein and its PD pathogenic mutations (A30P, A53T) the clumps with GFP were picked up at each passage. Clumps of hESCs were detached from the feeder layer by dispase and cultured in suspension with ES medium for 3 days. On day 4, formed embryoid bodies were transferred to NPC medium supplemented with bFGF. Three days'later, embryoid bodies were induced to adhere in the presence of 10% FBS. The next day, the medium was refreshed with neural induction medium supplemented with FGF8 and Shh instead of FGF2. Six days'after attachement the neural tube-like rosettes were picked up and cultured in neural induction medium supplemented with B-27, FGF8 and Shh for 6 days. Finally, the neurospheres were dissociated in accutase/trypsin (1:1), and plated on glass coverslips coated with laminin for terminal differentiation. The cells were feeded with NPC medium supplemented with BDNF, GDNF, FGF8, SHH, B27, AA, cAMP, and laminin, and half of the medium was changed every other day. Several days later perform immunocytochemistry to detect TH andα-synuclein.Results:The hESCs stably expressingα-synuclein and its PD associated mutations (A30P, A53T) can differentiate into neural precursor cells as well as dopaminergic neurons. Wild type and A53T-α-synuclein appears as large granular distribution in neural precursor cells as well as dopaminergic and non-dopaminergic neurons. which is different from the diffuse and punctate distribution in hESCs. But A30P-α-synuclein showed the same diffuse and punctate distribution as in hESCs.Conclusion:(1) There is no significant difference among the differentiation of hESCs stably expressing a-synuclein and its PD associated mutations (A30P, A53T) into neural precursor cells and dopaminergic neurons. (2) In the differentiated neural precursor cells as well as dopaminergic neuron wild type and A53T-α-synuclein appear as a similar cellular distribution which are different from A30P-α-synuclein.