Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Two critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes.</p> <p>Results</p> <p>We have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAs^CNTF), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDA^CNTFcells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAs^BMP did not exhibit pain syndromes.</p> <p>Conclusion</p> <p>Our results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.</p

Genetic Correction of Huntington's Disease Phenotypes in Induced Pluripotent Stem Cells

SummaryHuntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology

The Study of astrocyte development and function using rat embryonic spinal cord derived glial restricted precursors

Thesis (Ph. D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2008.The purpose of this thesis is to study the regulation of astrocyte differentiation and to study the function of astrocytes and their interaction with neurons. We isolated glial restricted precursors (GRP) from the rat embryonic neural tube and used this cell type to examine factors that influence the differentiation of astrocytes from GRPs. We demonstrated that endothelin3-mediated signaling was one modulator of astrocyte generation. Endothelin3 induced the generation of an astrocyte precursor cell but not of mature astrocytes from GRP cells. Additionally, endothelin3 blocked the generation of oligodendrocytes, the other potential cell fate of GRP cells. The suppression of the oligodendrocyte lineage pathway by endothelin3 was accompanied by the down-regulation of oligodendrocyte lineage transcription factors. One of these transcription factors, Olig2 was actively degraded by endothelin3 through a post-translational pathway. Olig2 was found to block astrocyte precursor induction when over-expressed in GRP cells. In addition to the effects of endothelin exposure, the intracellular redox state also affected astrocyte generation from GRP cells. More oxidized GRP cells differentiated into astrocytes more readily than reduced GRPs. Moreover, GRP cells dissected from different regions of the rat embryonic neural tube bad different redox states and demonstrated a varied potential to give rise to astrocytes. We also studied the properties of two populations of astrocytes derived from the same GRP cells in terms of the interactions of astrocytes with neurons. These two groups of astrocytes differed in the expression of Olig2, production of axon growth inhibitors, and the ability to support neuronal growth. Olig2 was also a critical regulator of astrocytic phenotype, since over-expression or knock-down of Olig2 changed the properties of either kind of astrocyte. Our work provides new insights into the early stages of astrocyte development and mechanisms underlying roles of astrocytes in injuries, which will enhance the ability to use astrocytes and their precursors in cell transplantation therapies for tissue repair

Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first exon of the gene Huntingtin (Htt). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells (NSCs) from HD-iPS cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained with β-III tubulin, calbindin and GABA but not DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs (from iPS or H9 NSCs). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD mechanisms and drug screening

Genomic Analysis Reveals Disruption of Striatal Neuronal Development and Therapeutic Targets in Human Huntington's Disease Neural Stem Cells.

We utilized induced pluripotent stem cells (iPSCs) derived from Huntington's disease (HD) patients as a human model of HD and determined that the disease phenotypes only manifest in the differentiated neural stem cell (NSC) stage, not in iPSCs. To understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls. Differential gene expression and pathway analysis pointed to transforming growth factor β (TGF-β) and netrin-1 as the top dysregulated pathways. Using data-driven gene coexpression network analysis, we identified seven distinct coexpression modules and focused on two that were correlated with changes in gene expression due to the CAG expansion. Our HD NSC model revealed the dysregulation of genes involved in neuronal development and the formation of the dorsal striatum. The striatal and neuronal networks disrupted could be modulated to correct HD phenotypes and provide therapeutic targets

Antibacterial activity of <i>Pyrrosia petiolosa</i> ethyl acetate extract against <i>Staphylococcus aureus</i> by decreasing <i>hla</i> and <i>sea</i> virulence genes

<p>The aim of this study was to explore the antibacterial activity of <i>Pyrrosia petiolosa</i> ethyl acetate extract (PPEAE) against <i>Staphylococcus aureus in vitro</i> and analyse its chemical components by gas chromatograph-mass spectrometry. The results of anti-microbial assay revealed that PPEAE had strong inhibitory activity against <i>S .aureus</i>, with MIC and MBC of 7.8 and 15.6 mg/mL, respectively. The transcriptional levels of <i>hla</i> and <i>sea</i> were reduced to 14.33 and 46.39% at the MIC compared to the control. Analysing test result exhibited that eugenol made a great contribution to antibacterial activity. This experiment indicated that PPEAE had prominent antibacterial activity against <i>S. aureus</i>.</p

FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16INK4a increase.

Neurodegenerative diseases (ND) have been linked to the critical process in aging-cellular senescence. However, the temporal dynamics of cellular senescence in ND conditions is unresolved. Here, we show senescence features develop in human Huntington's disease (HD) neural stem cells (NSCs) and medium spiny neurons (MSNs), including the increase of p16INK4a , a key inducer of cellular senescence. We found that HD NSCs reprogram the transcriptional targets of FOXO3, a major cell survival factor able to repress cell senescence, antagonizing p16INK4a expression via the FOXO3 repression of the transcriptional modulator ETS2. Additionally, p16INK4a promotes cellular senescence features in human HD NSCs and MSNs. These findings suggest that cellular senescence may develop during neuronal differentiation in HD and that the FOXO3-ETS2-p16INK4a axis may be part of molecular responses aimed at mitigating this phenomenon. Our studies identify neuronal differentiation with accelerated aging of neural progenitors and neurons as an alteration that could be linked to NDs

FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

International audienceNeurodegenerative diseases (ND) have been linked to the critical process in aging-cellular senescence. However, the temporal dynamics of cellular senescence in ND conditions is unresolved. Here, we show senescence features develop in human Huntington's disease (HD) neural stem cells (NSCs) and medium spiny neurons (MSNs), including the increase of p16INK4a , a key inducer of cellular senescence. We found that HD NSCs reprogram the transcriptional targets of FOXO3, a major cell survival factor able to repress cell senescence, antagonizing p16INK4a expression via the FOXO3 repression of the transcriptional modulator ETS2. Additionally, p16INK4a promotes cellular senescence features in human HD NSCs and MSNs. These findings suggest that cellular senescence may develop during neuronal differentiation in HD and that the FOXO3-ETS2-p16INK4a axis may be part of molecular responses aimed at mitigating this phenomenon. Our studies identify neuronal differentiation with accelerated aging of neural progenitors and neurons as an alteration that could be linked to NDs