Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells

Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers

Glial Progenitor-Like Phenotype in Low-Grade Glioma and Enhanced CD133-Expression and Neuronal Lineage Differentiation Potential in High-Grade Glioma

Background: While neurosphere-as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified. Methodology/Principal Findings: Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFR alpha, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRa, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype. Conclusions/Significance: This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas

Lithium Suppresses Astrogliogenesis by Neural Stem and Progenitor Cells by Inhibiting STAT3 Pathway Independently of Glycogen Synthase Kinase 3 Beta

Transplanted neural stem and progenitor cells (NSCs) produce mostly astrocytes in injured spinal cords. Lithium stimulates neurogenesis by inhibiting GSK3b (glycogen synthetase kinase 3-beta) and increasing WNT/beta catenin. Lithium suppresses astrogliogenesis but the mechanisms were unclear. We cultured NSCs from subventricular zone of neonatal rats and showed that lithium reduced NSC production of astrocytes as well as proliferation of glia restricted progenitor (GRP) cells. Lithium strongly inhibited STAT3 (signal transducer and activator of transcription 3) activation, a messenger system known to promote astrogliogenesis and cancer. Lithium abolished STAT3 activation and astrogliogenesis induced by a STAT3 agonist AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), suggesting that lithium suppresses astrogliogenesis by inhibiting STAT3. GSK3β inhibition either by a specific GSK3β inhibitor SB216763 or overexpression of GID5-6 (GSK3β Interaction Domain aa380 to 404) did not suppress astrogliogenesis and GRP proliferation. GSK3β inhibition also did not suppress STAT3 activation. Together, these results indicate that lithium inhibits astrogliogenesis through non-GSK3β-mediated inhibition of STAT. Lithium may increase efficacy of NSC transplants by increasing neurogenesis and reducing astrogliogenesis. Our results also may explain the strong safety record of lithium treatment of manic depression. Millions of people take high-dose (>1 gram/day) lithium carbonate for a lifetime. GSK3b inhibition increases WNT/beta catenin, associated with colon and other cancers. STAT3 inhibition may reduce risk for cancer

Loss of ATM Induces Glial Cell Dysfunction in a Novel Murine Model of Ataxia-telangiectasia

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, Program in Genetics, 2016.Ataxia-telangiectasia (A-T) is a rare, recessive, pediatric disorder that is primarily characterized by cerebellar degeneration of Purkinje neurons and results from mutations in the Ataxia-telangiectasia Mutated (ATM) gene. Patients exhibit extreme sensitivity to ionizing radiation, immunodeficiency, an increased risk for malignancy, and increased oxidative stress. In order to study the effects of true loss of ATM, our lab has generated an inducible A-T mouse model, AtmMmpl, with a novel N-terminal mutation. The mutant mice show decreased levels of peripheral T and B cell populations, have a low incidence of thymic lymphoma, and do not show evidence of increased oxidative stress. Our analyses of these phenotypes in two different strains of AtmAwb mice indicate that the background genetics may play a role in modulating the disease manifestation. Despite a mild motor phenotype, the development and morphology of the cerebellum is normal, with no overt change in Purkinje cell number or morphology. Analysis of other critical cell types within the cerebellum also did not reveal any significant changes. Preliminary RNAseq analysis suggests that there may be compensatory mechanisms employed by our mice that protect against the consequences of loss of ATM. However, we found evidence of a cortical white matter defect in our year old mice, which is reminiscent of reports in A-T patients. In order to test if the loss of ATM results in an underlying defect that would be exacerbated upon an insult, we exposed our murine model to cuprizone, a common model for studying demyelination. We hypothesized that the loss of ATM in the glial cell populations, such as the oligodendrocytes and oligodendrocyte precursor cells, would make them more vulnerable and less capable of responding to a neurotoxic insult. We found that the A-T mutant mice are more sensitive to cuprizone-induced demyelination, showing increased oligodendrocyte cell death, and have an increased inflammatory environment following treatment, but are capable of repair. These data demonstrate that there is a novel glial cell dysfunction that is induced by the loss of ATM

Disruption of ATM function and divergent phenotypes in two mouse models of ataxia-telangiectasia

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2014.Ataxia-telangiectasia (A-T) is a rare, systemic disease characterized by immunodeficiency, increased incidence of cancer, and progressive cerebellar neurodegeneration resulting in loss of gross motor control. Individuals with A-T harbor homozygous recessive mutations in the ATM gene, resulting in truncations, inactivation, or loss of ATM protein. Despite common underlying genetic defects, significant heterogeneity exists in the manifestation of symptoms. As one of ATM’s primary functions involves regulating the cellular response to DNA damage, the symptoms associated with A-T are thought to result from the inability of cells to repair DNA breaks occurring during development (T- and B-cell receptors) or regulate proliferation and maintain viability after cytogenetic insults (cancer, premature senescence). Although ATM plays an important role in regulating the cell cycle and maintaining genomic integrity in post-mitotic neurons, this alone cannot explain the differential loss of neuronal populations in A-T. The response of individual neurons to injury (repair/survival or death) depends on a variety of factors, including the endogenous and exogenous levels of oxidative stress. As astrocytes are the primary regulators of oxidative stress in the brain, we hypothesized that Atm mutant mouse astrocytes modulate the survival of cerebellar neurons in A-T. Atm mutant astrocytes were impaired in their ability to support neuronal survival due to decreased glutathione synthesis and secretion that resulted from reduced expression of the cystine/glutamate antiporter subunit xCT. These results indicate that astrocyte dysfunction plays an important role in A-T and that restoration of astrocytic anti-oxidant support can improve neuronal survival. Despite the utility of current Atm mutant mice for studying the A-T, all lack the severe neurodegeneration associated with the disease. As these mice contain inactivating or truncation mutations, we hypothesized that the expression of residual Atm protein may attenuate neurodegeneration. To address this, we created novel Atm mutant mice containing a 5’ null mutation. These animals showed an impaired DNA damage response, immunodeficiency and cerebellar defects but a surprising lack of cancer. These results provide new insights into role of Atm in development and tumorigenesis and highlight the need for a better understanding of the cellular and genetic factors that may modulate the disease

Human Herpesvirus 6A Latency Gene U94A Impairs Motility and Maturation in Central Nervous System Cell Types: Implications for Neurodegenerative Disease

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Neuroscience Graduate Program, 2020.Many neurodegenerative diseases have a multifactorial etiology and variable course of progression that cannot be explained by current models. Neurotropic viruses have long been suggested to play a role in these diseases, although their exact contributions remain unclear. Human herpesvirus 6A (HHV-6A) is one of the most common viruses detected in the adult brain, and has been clinically associated with multiple sclerosis (MS), and, more recently, Alzheimer’s disease (AD). HHV-6A is a ubiquitous viral pathogen capable of infecting glia and neurons. Primary infection in childhood is followed by the induction of latency, characterized by expression of the U94A viral transcript in the absence of viral replication. Our work is the first to examine the effects of this common viral gene on cells of the central nervous system. We found that U94A expression inhibits the migration and maturation of human oligodendrocyte precursor cells (OPCs) without affecting their viability, a phenotype that may contribute to the failure of remyelination seen in many patients with MS. Large-scale transcriptomics and proteomics analyses indicate that U94A expression in OPCs alters the expression of genes involved in cytoskeletal regulation and in cellular interactions with the extracellular matrix, while preliminary biochemical analyses suggest that U94A may exert its functions by interacting with nucleosomes and with myosin motors. As HHV-6A seems to be significantly associated with early AD pathology, we extended our initial analysis of U94A in OPCs to cytoskeletal abnormalities in neurons. We found that U94A expression inhibits morphological maturation in human cortical neurons. Given that morphological abnormalities are known to precede synapse loss and cognitive impairment in AD patients, we hypothesize that U94A expression in neurons renders them more susceptible to dysfunction and degeneration. Our work suggests that the persistent presence of HHV-6A-associated proteins establishes a state of vulnerability that can contribute to disease progression in MS and AD. We propose this virus as a unique human factor to consider in the translation of therapies from animal models to human patients

Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses (HVs) have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system (CNS). The ability of HVs to enter a state of latency, a defining characteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease (NDD) pathology by highlighting two prominent members of the HV family, herpes simplex virus 1 (HSV-1) and human herpesvirus 6 (HHV-6). We (i) introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then (ii) review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer's disease (AD) and multiple sclerosis (MS), respectively. We then (iii) highlight and discuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we (iv) discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs