Turhan Coksaygan

β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche

IInteractions between laminins and integrin receptors hold neural stem cells in place at the ventricular surface of embryonic brain. Transient disruption leads to abnormal stem cell divisions and permanent cortical malformation.

Evidence that nucleocytoplasmic Olig2 translocation mediates brain-injury-induced differentiation of glial precursors to astrocytes

The mechanisms by which neural and glial progenitor cells in the adult brain respond to tissue injury are unknown. We studied the responses of these cells to stab wound injury in rats and in two transgenic mouse models in which Y/GFP is driven either by Sox2 (a neural stem cell marker) or by Tα‐1 (which marks newly born neurons). The response of neural progenitors was low in all nonneurogenic regions, and no neurogenesis occurred at the injury site. Glial progenitors expressing Olig2 and NG2 showed the greatest response. The appearance of these progenitors preceded the appearance of reactive astrocytes. Surprisingly, we found evidence of the translocation of the transcription factor Olig2 into cytoplasm in the first week after injury, a mechanism that is known to mediate the differentiation of astrocytes during brain development. Translocation of Olig2, down‐regulation of NG2, and increased glial fibrillary acidic protein expression were recapitulated in vitro after exposure of glial progenitors to serum components or bone morphogentic protein by up‐regulation of Notch‐1. The glial differentiation and Olig2 translocation could be blocked by inhibition of Notch‐1 with the γ‐secretase inhibitor DAPT. Together, these data indicate that the prompt maturation of numerous Olig2+ glial progenitors to astrocytes underlies the repair process after a traumatic injury. In contrast, neural stem cells and neuronal progenitor cells appear to play only a minor role in the injured adult CNS.

Increased Dentate Neurogenesis After Grafting of Glial Restricted Progenitors or Neural Stem Cells in the Aging Hippocampus

Neurogenesis in the dentate gyrus (DG) declines severely by middle age, potentially because of age‐related changes in the DG microenvironment. We hypothesize that providing fresh glial restricted progenitors (GRPs) or neural stem cells (NSCs) to the aging hippocampus via grafting enriches the DG microenvironment and thereby stimulates the production of new granule cells from endogenous NSCs. The GRPs isolated from the spinal cords of embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase gene and NSCs isolated from embryonic day 9 caudal neural tubes of Sox‐2:EGFP transgenic mice were expanded in vitro and grafted into the hippocampi of middle‐aged (12 months old) F344 rats. Both types of grafts survived well, and grafted NSCs in addition migrated to all layers of the hippocampus. Phenotypic characterization revealed that both GRPs and NSCs differentiated predominantly into astrocytes and oligodendrocytic progenitors. Neuronal differentiation of graft‐derived cells was mostly absent except in the dentate subgranular zone (SGZ), where some of the migrated NSCs but not GRPs differentiated into neurons. Analyses of the numbers of newly born neurons in the DG using 5′‐bromodeoxyuridine and/or doublecortin assays, however, demonstrated considerably increased dentate neurogenesis in animals receiving grafts of GRPs or NSCs in comparison with both naïve controls and animals receiving sham‐grafting surgery. Thus, both GRPs and NSCs survive well, differentiate predominantly into glia, and stimulate the endogenous NSCs in the SGZ to produce more new dentate granule cells following grafting into the aging hippocampus. Grafting of GRPs or NSCs therefore provides an attractive approach for improving neurogenesis in the aging hippocampus.

Cytoplasmic translocation of Olig2 in adult glial progenitors marks the generation of reactive astrocytes following autoimmune inflammation.

The injury response in the brain involves complex interplay between neural and immune components. Following inflammatory insults to the adult CNS, formation of an astroglial scar often impedes functional repair. Glial progenitor cells expressing the nuclear transcription factor Olig2 possibly generate astrocytes in response to various types of injuries; however, the mechanisms underlying this differentiation are unclear. In a model of immune-mediated injury (MOG(35-55)-experimental autoimmune encephalomyelitis), we show that the conversion from progenitor to reactive astrocyte is marked by the translocation of Olig2 into the cytoplasm. Evidence of this process is found for months after disease initiation in the absence of new inflammatory infiltrates. A proportion of cells with cytoplasmic Olig2 was found to express NG2 or Nkx2.2, but only Nkx2.2 was occasionally retained by GFAP+ cells. We further show that differentiation to astrocytes is induced in glial progenitors in vitro through exposure to the pro-inflammatory cytokine IFN-gamma, but not to TNF-alpha. Together, these data ascribe a pivotal role to Olig2+ glial precursor cells in the adult CNS, linking autoimmune inflammation and glial scar formation.

Adverse Effects of Antenatal Glucocorticoids on Cerebral Myelination in Sheep

OBJECTIVE: To determine in fetal sheep the effect of betamethasone on myelination in relation to stage of myelination, number of treatment courses, dose, and route of administration. METHODS: Fetal expression of myelin basic protein (MBP), a marker of mature oligodendrocytes and myelin, was determined between 0.27 and 0.93 gestation. Short-term betamethasone effects were examined 24 hours after one maternal intramuscular treatment course (weight adjusted to equal the clinical dose of 2×8 mg betamethasone to a 70-kg woman) at 0.63, 0.75, and 0.87 gestation or after continuous 48-hour fetal intravenous infusion at 0.75 and 0.87 gestation. Lasting effects were examined 20 days after one and two treatment courses weight-adapted to the clinical dose of 2×8 mg or 2×12 mg betamethasone at 0.75 gestation. RESULTS: Myelin basic protein immunoreactivity was first detected in the internal capsule at 0.53 gestation, followed by the centrum semiovale, the superficial white matter, and corpus callosum at 0.63 gestation. Within 24 hours after treatment, betamethasone reduced the number of mature oligodendrocytes and MBP immunoreactivity. The effect decreased with gestational age. Maternal and fetal betamethasone administration had similar effects. Loss of MBP immunoreactivity was not reversed 20 days after two treatment courses, independent of dose. CONCLUSION: Betamethasone-induced delayed cerebral myelination is dependent on the stage of brain development in sheep. Betamethasone-related disturbances in myelination and any potential contribution to childhood behavior deficits need to be confirmed in clinical studies.

Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism

During development of the mammalian cerebral cortex neural stem cells (NSC) first generate neurons and subsequently produce glial cells. The mechanism(s) responsible for this developmental shift from neurogenesis to gliogenesis is unknown. Brain‐derived neurotrophic factor (BDNF) is believed to play important roles in the development of the mammalian cerebral cortex; it enhances neurogenesis and promotes the differentiation and survival of newly generated neurons. Here, we provide evidence that a truncated form of the BDNF receptor tyrosine kinase B (trkB‐t) plays a pivotal role in directing embryonic mouse cortical NSC to a glial cell fate. Expression of trkB‐t promotes differentiation of NSC toward astrocytes while inhibiting neurogenesis both in cell culture and in vivo. The mechanism by which trkB‐t induces astrocyte genesis is not simply the result of inhibition of full‐length receptor with intrinsic tyrosine kinase activity signaling. Instead, binding of BDNF to trkB‐t activates a signaling pathway (involving a G‐protein and protein kinase C) that induced NSC to become glial progenitors and astrocytes. Thus, the increased expression of trkB‐t in the embryonic cerebral cortex that occurs coincident with astrocyte production plays a pivotal role in the developmental transition from neurogenesis to gliogenesis. Our findings suggest a mechanism by which a single factor (BDNF) regulates the production of the two major cell types in the mammalian cerebral cortex.

Neurogenesis in Tα-1 tubulin transgenic mice during development and after injury

Talpha-1 tubulin promoter-driven EYFP expression is seen in murine neurons born as early as E9.5. Double labeling with markers for stem cells (Sox 1, Sox 2, nestin), glial progenitors (S100beta, NG2, Olig2), and neuronal progenitors (doublecortin, betaIII-tubulin, PSA-NCAM) show that Talpha-1 tubulin expression is limited to early born neurons. BrdU uptake and double labeling with neuronal progenitor markers in vivo and in vitro show that EYFP-expressing cells are postmitotic and Talpha-1 tubulin EYFP precedes the expression of MAP-2 and NeuN, and follows the expression of PSA-NCAM, doublecortin (Dcx), and betaIII-tubulin. Talpha-1 tubulin promoter-driven EYFP expression is transient and disappears in most neurons by P0. Persistent EYFP expression is mainly limited to scattered cells in the subventricular zone (SVZ), rostral migratory stream, and hippocampus. However, there are some areas that continue to express Talpha-1 tubulin in the adult without apparent neurogenesis. The number of EYFP-expressing cells declines with age indicating that Talpha-1 tubulin accurately identifies early born postmitotic neurons throughout development but less clearly in the adult. Assessment of neurogenesis after stab wound injuries in the cortex, cerebellum and spinal cord of adult animals shows no neurogenesis in most areas with an increase in BrdU incorporation in glial and other non neuronal populations. An up-regulation of Talpha-1 tubulin can be seen in certain areas unaccompanied by new neurogenesis. Our results suggest that even if stem cells proliferate their ability to generate neurons is limited and caution is warranted in attributing increased BrdU incorporation to stem cells or cells fated to be neurons even in neurogenic areas.

LRP1 Protects the Vasculature by Regulating Levels of Connective Tissue Growth Factor and HtrA1

Objective—Low-density lipoprotein receptor–related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1−/−) on a low-density lipoprotein receptor–deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. Approach and Results—We found smLRP1−/− mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1−/− vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. Conclusions—Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy.

Niemann–Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-β-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.

The Protective Effect of Glibenclamide in a Model of Hemorrhagic Encephalopathy of Prematurity

We studied a model of hemorrhagic encephalopathy of prematurity (EP) that closely recapitulates findings in humans with hemorrhagic EP. This model involves tandem insults of 20 min intrauterine ischemia (IUI) plus an episode of elevated venous pressure induced by intraperitoneal glycerol on post-natal day (P) 0. We examined Sur1 expression, which is upregulated after focal ischemia but has not been studied after brief global ischemia including IUI. We found that 20 min IUI resulted in robust upregulation of Sur1 in periventricular microvessels and tissues. We studied tandem insult pups from untreated or vehicle-treated dams (TI-CTR), and tandem insult pups from dams administered a low-dose, non-hypoglycemogenic infusion of the Sur1 blocker, glibenclamide, for 1 week after IUI (TI-GLIB). Compared to pups from the TI-CTR group, pups from the TI-GLIB group had significantly fewer and less severe hemorrhages on P1, performed significantly better on the beam walk and accelerating Rotarod on P35 and in tests of thigmotaxis and rapid learning on P35–49, and had significantly greater body and brain weights at P52. We conclude that low-dose glibenclamide administered to the mother at the end of pregnancy protects pups subjected to IUI from post-natal events of elevated venous pressure and its consequences.