Neuroprotective Characterization Of KCNB1 Cleavage By BACE2 And Differentiation Patterns Of Human Induced Pluripotent Stem Cells Into Cortical Projection Neurons

PART I Neuroprotective Characterization of KCNB1 Cleavage by BACE2Alzheimer’s disease (AD) is the most common neurodegenerative disease, accounting for 60% to 70% of cases of dementia. It has been associated with varieties factors, such as the genetic background, P-amyloid, oxidative stress, inflammation and insulin resistance. The molecular mechanisms and hypotheses of AD are incredibly complexed. The key event leading to AD is the formation of a peptide known as amyloid beta (Aβ). Aβ peptide is generated following the sequential cleavage of amyloid precursor protein (APP) by β-and y-secretase in the amyloidogenic pathway. β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartic protease, which is widely expressed in various tissues in human, especially highly expressed in human AD brain. Recently, the homology β-site APP cleaving enzyme 2 (BACE2) has been reported. Researches found that BACE2 was lowly expressed in neurons of health person, but highly expressed in the neurons of AD brains. One study has identified a new functional role for BACE2 as a potent Ab degrading protease. Thus, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.KCNB1, also known as kv2.1, is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium channel in mammalian central neurons. It has six transmembrane segments S1-S6 arranged in modular voltage-sensing (S1-S4) and pore-forming (S5-S6) domains, and extensive N-and C-terminal cytoplasmic domains. The N terminus contains the tetramerization or T1 domain that mediates sub unit tetramerization. The intracellular N-and C-terminal cytoplasmic domains include 75 serine,36 threonine and 13 tyrosine residues, which could be modified by the intracellular protein kinases and phosphatases. KCNB1 regulates intrinsic excitability during periods of high-frequency firing in pyramidal cells or tonic firing in sympathetic neurons. Besides, KCNB1 also plays a role in the regulation of apoptosis. The increased membrane expression of KCNB1 in cortical neurons promotes neuronal apoptosis. Silencing or transiently expression of a dominant-negative (DN) truncated mutant of KCNB1 could efficiently prevent neuronal apoptosis. Moreover, KCNB1 is also the target of reactive oxygen species. Previous studies have demonstrated KCNB1 was oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of AD. KCNB1 oxidation acts to enhance apoptosis, whereas KCNB1-C73A and KCNB1-C710A, two variants resistant to oxidative modification, are cytoprotective.Our study here showed KCNB1 was a novel substrate of BACE2. BACE2 cleaved KCNB1 after Met375, Lys716 and Tyr768 and oxidative stress, such as Aβ1-42, blocked BACE2 cleavage. Moreover, by cleavage, BACE2 disrupted KCNB1 clustering on cell membrane, decreased Ik of KCNB1 and led to a hyperpolarizing shift. Finally, we demonstrated that the truncated fragments, KCNB1-1-375, KCNB1-1-716 and KCNB1-1-768, alleviated neuron apoptosis induced by 2,2’-dithiod ipyrid ine.PART II Differentiation Patterns of Human Induced Pluripotent Stem Cells into Cortical Projection NeuronsThe cerebral cortex of mammalian is consist of six-layer, L1-L6, developing from inside to the outside and regulating by a series of intricate mechanism. Many mutations of coding gene associated with the cortical development can result in the developmental diseases of the brain. So far, the most common model for the study of molecular pathogenesis of these diseases are the site-directed mutagenesis mouse models. But the mouse models are not only time-consuming but also have mass distinctions with human.In 2006, Yamanaka et. al, has reported that the forced expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, could reprogram fibroblasts to induced pluripotent stem cells (iPS cells). The advent of iPS cells provided an important tool for the study of human neurodegenerative and neurodevelopmental diseases in live neurons in a controlled environment. Up to now, many disease-specific iPS cell lines have been built (examples AD, PD, ALS, SMA). These iPS cell lines have contribute a lot in elucidating the pathogenesis of disease. Despite the widespread use of iPS in the research of neurology, the differentiation patterns of iPS into cortical projection neurons are still uncertain.Our current research clarified the differentiation patterns of iPS into cortical projection neurons by identifying the neuron types at different differentiational stages using immune staining. We established iPS cells lines by Episomal plasmid nuclear transfection method. The in vitro embryo bodies and in vivo teratomas analysis was performed to detected the potential of differentiating into three germ layers. We differentiated iPS cells into cortical projection neurons using small molecules, such as LDN 193189, XAV 939 and SB 431542. Immunostaing was used to determin the neural types at different stages of neural differentiation and electrophysiological recording was performed to evaluate the maturity of neurons. Our results showed iPS clones expressed embryonic stem cell markers, such as OCT4、SOX2、Nanog、SSEA4 and TRA1-60. In vitro embryo and in vivo teratoma analysis demonstrated iPS cells could differentiate into three germ layers. After 12 days of neural induction, the iPS cells highly expressed FOXG1、PAX6、OTX1 and SOX1, which were known as markers of neural precursor cells. On Day 35, the neurons gradually became mature, and only TBR1+neurons in L6 and SP could be detected. However, the cortical projection neurons in L5-L2 could be detected on Day 80. Electrophysiological recording indicated that as neurons mature, the action potential progress from firing a short burst of action potentials in response to current injection (Day 35) to sustained action potential firing (Day 80).Thus, after 80 days of neural induction, the iPS cells were differentiated into cortical projection neurons in an "inside-outside" fashion, which is similar to that in vivo cortical neurons development. This established methodology provides an ideal research model for the research of neurological diseases.