Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

peer reviewedIn addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke

ES細胞培養系に於ける外因性シグナルの添加時期及び組合わせ作用による腹側終脳組織の領域特異化

京都大学0048新制・課程博士博士(医学)甲第15971号医博第3556号新制||医||986(附属図書館)28550京都大学大学院医学研究科医学専攻(主査)教授 中辻 憲夫, 教授 金子 武嗣, 教授 中畑 龍俊学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

<div><p>In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.</p> </div

Multi-layered cortical NE on day 12.

<p>(A–B) Day-12 aggregates carrying continuous Foxg1::venus⁺ NE epithelia. (A) Fluorescent image of a Foxg1::venus⁺ aggregate. (B) Immunostaining for Foxg1. (C–H) Layered formation in continuous Foxg1::venus⁺ NE epithelia. (C–E) Cortical progenitors (Pax6⁺ and Emx1⁺) form a zone on the apical side of Foxg1::venus⁺ NE. (F) Immunostaining for Ngn2⁺ cells located in the basal part of the progenitor zone. A majority of basal progenitors (Tbr2⁺) were found outside of the Ngn2⁺ cell zone. (G–H) Early cortical plate neurons (Tbr1⁺, Ctip2⁺) were found on the basal side of Foxg1::venus⁺ NE epithelia. (I–M) Differential location of layer-specific cortical neurons in Foxg1::venus⁺ NE epithelia. (I–K) Immunostaining for Ctip2 (I–J) and Tbr1 (K). (L–M) Immunostaining for Brn2 (L) and Cux1 (M). Dashed lines indicate the apical and basal borders of NE. Scale bars, 200 µm (A–B); 100 µm (C–M).</p

Formation and spatial arrangement of non-cortical telencephalic tissues.

<p>(A) Expression pattern of markers for cortex, cortical hem, and choroid plexus on embryonic day 12 (E12). (B) Hem-derived Cajal-Retzius cells expressing p73 on E12. (C–D) The hem-like tissues (Lmx1a⁺, Otx2⁺) formed adjacent to cortical tissues (Foxg1::venus⁺) in culture. The hem-like tissues also contained p73⁺ cells. (E) Schematic for SFEBq/gfCDM+IwL culture with Shh treatment. (F–H) <i>In vivo</i> expression of pallial and subpallial markers on E12. Cortical markers (Pax6, Ngn2, Tbr1), LGE markers (Gsh2, Dlx2, Mash1, Gad65), and MGE markers (Nkx2.1, Dlx2, Mash1, Gad65). (I) Schematic of the marker expression pattern. (J–M) Shh treatment induced the formation of subpallial tissues. LGE tissues (Gsh2⁺, Dlx2⁺, Mash1⁺) were located between cortical tissues (Pax6⁺, Ngn2⁺, Tbr1⁺) and MGE tissues (Nkx2.1⁺, Dlx2⁺, Mash1⁺). Arrowheads indicate a transition between pallial and subpallial tissues. (N) Expression of Gsh2::venus in thickened NE tissue on day 14. (O–Q) Immunostaing of Gsh2::venus⁺ NE on day 14. Immunostaining for Gsh2 (O), Mash1 (P) and Gad67/Dlx2 (Q). chp, choroid plexus; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; PSB, pallial-subpallial boundary; Th, thalamus. Scale bars, 100 µm (A–D,J–M,O–Q); 200 µm (F–H,N).</p

Multi-layered cortical NE on day 15.

<p>(A) Day-15 aggregate carrying continuous Foxg1::venus⁺ NE epithelia. Immunostaining for GFP. (B–G) Layer formation without the inside-out pattern. (B–C) Cortical progenitors (Foxg1::venus⁺/Pax6⁺) on the apical side. (D) Ngn2⁺ cells were located in a slightly deeper zone. Basal progenitors (Tbr2⁺) were outside of the Ngn2⁺ zone (E–G) Early cortical plate neurons (Tbr1⁺, Ctip2⁺) occupied the basal zone of continuous Foxg1::venus⁺ NE epithelia (E–F), while late cortical plate neurons (Brn2⁺, Cux1⁺) stayed on the apical side. (H) A schematic of cortical layer formation <i>in vitro</i> on day 15. Dashed lines indicate the apical or basal borders of NE. CR, Cajal-Retzius cell. Scale bars, 100 µm.</p

Use of “MGE Enhancers” for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

<div><p>The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6⁺ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6⁺ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [<i>422</i> (<i>DlxI12b</i>), <i>Lhx6</i>, <i>692</i>, <i>1056</i>, and <i>1538</i>] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers <i>DlxI12b</i>, <i>692</i>, and <i>1538</i> are active in Lhx6-GFP⁺ cells, while enhancer <i>1056</i> is active in Olig2⁺ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.</p></div