Angela Jane Roskams
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin.Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.
The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.
Master's Student Supervision (2010 - 2018)
The developing mammalian spinal cord can generate all required cell types; in the adult, there exists a subpopulation of progenitors able to generate multiple cell types. The ependymal cell (EC) layer of the central canal (CC) of the adult spinal cord is a neural stem cell niche possessing the potential to self-renew and differentiate into astrocytes and oligodendrocytes in vivo and in vitro, and neurons in vitro. However, the differences between a developmental EC and an adult EC are not well established. Further, it is unknown whether ECs have equivalent stem/progenitor abilities, and whether certain subpopulations may be better targets for regeneration. Multiple Sclerosis (MS) is a debilitating disease characterized by areas of chronically demyelinated CNS, and an attractive target for regenerative therapy. Little is known about why endogenous regenerative programmes in MS are unsuccessful. Using in-situ hybridization (ISH) data from the Allen Spinal Cord Atlas and immunofluorescent detection of antigen in mice, we revealed temporal and spatial segmentation of ECs expressing distinct groups of genes. The ages analyzed (postnatal day 4 and 56) are relevant to the mechanisms of neurogenesis and gliogenesis, as the spinal cord is capable of regaining greater functionality following injury at P4 than at P56. Gene ontology analysis suggests different regions of the CC may contribute in different ways to spinal cord repair. Furthermore, we found the EC niche experiences altered levels of proliferation after two different types of insult, SCI and demyelinating injury in mice. We then used MS spinal cord to test what extent such repair from demyelination takes place. We found that remyelination occurred, but was inadequate and highly variable both between and within patients. Abnormal myelinating cells of a unique phenotype were found in the CNS and PNS; the extent and prevalence of peripheral involvement has not been previously reported. Further work is needed to determine whether the human EC layer could be targeted to enhance remyelination. Overall, these results reveal novel cell phenotypes engaged in repair in mouse and human spinal cord that may serve a basis for future therapeutic intervention.
Myelin is important for axon maintenance and survival, as well as for saltatory conduction of nerve impulses. Consequently, loss of myelin after spinal cord injury or in demyelinating diseases such as Multiple Sclerosis (MS) results in dysfunction of nerve impulse propagation and progressive axonal damage and cell death. Endogenous remyelination can occur in response to MS and is mainly mediated by oligodendrocyte precursor cell-derived oligodendrocytes, but Schwann cells (SCs) can also participate. SC remyelination has been documented in spinal cord lesions following traumatic spinal cord injury in humans, and in animal models of demyelination such as lysolecithin-induced demyelination, but endogenous remyelination by SCs in the context of MS has not been as well studied.In the present study we used immuno-fluorescent detection to analyze the expression of brain lipid binding protein (BLBP) and peripheral myelin protein zero (P0) in MS and non-MS human spinal cord as well as in the lysolecithin-demyelinated mouse spinal cord. BLBP (also known as Fatty Acid Binding Protein 7) is a nervous system-specific fatty acid binding protein. In the context of the present study, BLBP is important because it has been previously used to identify spinal cord radial glia (RG) in the developing and adult mouse spinal cord. BLBP is also expressed by SC precursors, and by immature SCs before they differentiate into myelinating SCs. We investigated whether, like the mouse spinal cord, the aged human spinal cord preserves a population of BLBP+ spinal cord RG. We found that (1) in contrast to the mouse spinal cord RG, human spinal cord RG do not express BLBP; (2) unlike the mouse, some subpopulations human myelinating SCs express BLBP in the PNS of MS (and some non-MS) cases; (3) in the MS spinal cord, BLBP-positive SCs extensively myelinate axons in large GFAP-rich areas; (4) and that BLBP is more readily detected in uncompacted myelin sheaths.Collectively these data provide evidence of robust SC remyelination in the human spinal cord beyond what has been previously reported, and highlight BLBP as a developmentally regulated protein whose expression is significantly different between mouse and human spinal cord RG and SCs
SPARC (Secreted Protein Acidic and Rich in Cysteine), a secreted glycoprotein, regulates proliferation, migration and differentiation. SPARC is highly expressed in glia and blood vessels during CNS development. SPARC expression is maintained in tissues undergoing rapid turnover and its expression is highly upregulated during injury or disease. SPARC’s modulatory activity in glia and endothelia during injury lead us to investigate the role of SPARC in an animal model of CNS inflammation and demyelination with known BBB dysfunction: Experimental Autoimmune Encephalomyelitis (EAE). We discovered that, in the spinal cord, SPARC is expressed and localized to developing endothelia and radial glia but is down-regulated and retained in specific subpopulations of glia in the adult spinal cord. During the repair response of EAE, CNS glia and endothelia recapitulate their developmental SPARC expression. Furthermore, in the absence of SPARC, EAE onset is delayed even though there is increased blood-brain barrier (BBB) permeability. We provide evidence that SPARC may play a role in neuro-immune and endothelial cross-talk during the repair response following EAE.
The human corticospinal tract (CST) is responsible for coordinated voluntary movement and it contains descending afferent inputs involved in autonomic control and gating of spinal reflexes. After spinal cord injury (SCI), damage to the CST causes degeneration of axons and can result in major motor impairments. The CST is especially lacking in its capacity to regenerate after injury. In the current study, we harvested the cortices of postnatal day 8 Thy1YFP16JRS mice, which express YFP in layer five projection neurons, which also express CST transcription factors Ctip2 and Otx1 in vitro. We applied Histone deacetylase (HDAC) inhibitors (Trichostain A [TSA] and Tubastatin A) to the mixed neuron culture and assessed survival and neurite outgrowth of YFP positive CST neurons. TSA treatment increased the number of primary neurites per neuron and the number of branch points exhibited by YFP positive CST neurons. Application of either TSA or Tubastatin A, promoted YFP positive CST neurite outgrowth in baseline media as well as in the presence of the neurotrophin 3 (NT3) and cilliary neurotrophic factor (CNTF), compared to the appropriate controls. Taken together, the application of HDAC inhibitors to postnatal corticospinal neurons can promote neurite outgrowth, branching and an increase in the number of primary neurites when grown in baseline media.