Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.

Large numbers of people suffer from nervous system injuries and neurodegenerative diseases each year, with little success in regaining lost neural functions. Attempts to successfully regenerate nervous tissue in the mammalian Central Nervous System have meet with limited success. Simpler models have thus been useful in determining conserved mechanisms in the enhancement of neural regeneration. One such mechanism is intracellular calcium signaling. We used <italic>Caenorhabditis elegans</italic> as a model system to study the effects of optogenetic stimulation on regeneration. Using a femtosecond laser we cut individual <italic>C. elegans</italic> axons <italic>in vivo</italic> and then periodically stimulated the neurons by activating the genetically encoded light activated channel, Channelrodopsin-2. We found that periodic photo-activation could increase regeneration over 24h by at least 31%. We repeated these experiments with dantrolene treatment and in <italic>unc-68(e540)</italic> mutants to assess the effects from a lack of internal calcium ion signaling in these worms. In both cases, we found a complete suppression of stimulated regeneration when calcium signaling was blocked. This indicates that intracellular calcium ion signaling is crucial in the initiation of neural regeneration in the first 24 hours and mediates the enhanced outgrowth we observe with periodic photo-activation. The importance of intracellular calcium ion signaling can lead to further studies to enhance the stimulation of neural regeneration, and improve therapies for patients with neural damage and loss of neural functions.

The use of highly active antiretroviral therapy (HAART) has significantly decreased the mortality rate of HIV-1 patients, however the increased survival has led to the development of complications associated with the persistence of the viral infection. Nearly half of HIV-1-infected individuals develop HIV-associated neurocognitive disorders (HAND) as the effects of the chronic infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxicity of HIV-1 has largely been attributed to the inflammation caused by viral replication and the altered signaling of astrocytes, microglia, and macrophages. Although HAART has improved the control of viral replication, the effects from inflammation remain a concern, particularly those of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α has been a therapeutic target for other diseases associated with chronic inflammation, such as rheumatoid arthritis, but emerging evidence has suggested that TNF-α signaling can have a dual role, especially in the CNS, proving the complexity in the modulation of the TNF-α pathway. Although the detrimental effects of TNF-α have been well-characterized, we lack a complete understanding of the beneficial role of TNF-α. TNF-α signaling has largely been considered to be neurotoxic but has been able to regulate neurite outgrowth in the context of neural development. Since TNF-α is upregulated in various neurodegenerative conditions, we considered potential outcomes of TNF-α on neurite outgrowth following injury. Initially, most would assume that TNF-α would prevent neurite outgrowth as apoptosis is a common outcome of TNF-α-induced signaling. If TNF-α signaling strictly prevents neurite outgrowth, anti-TNFα therapies could be considered to reverse this effect. However, upon induced injury, we observed an increase in neurite regrowth following induced injury in human primary fetal neurons, demonstrating a strong need for a deeper understanding of this dual role of TNF-α. Anti-TNF-α therapies have been considered for HIV-1-infected patients to reduce the chronic inflammation, however inhibiting TNF-α signaling could have side-effects that could prevent neuronal recovery from HIV-1 effects. Targeting pathways downstream of TNF-α signaling would be more advantageous to mediate the beneficial role of TNF-α in the CNS. We investigated the transcriptional effects of TNF-α treatment on neurons to uncover a potential pathway to promote neurite outgrowth. One pathway we have discovered to be beneficial in primary human fetal neurons is TNF-α-induced Ephrin B2 upregulation. Ephrin B2 (EphB2) receptors are important mediators of neuronal development and synaptic plasticity, however little has been established in regards to their role in HIV and inflammation, particularly in the CNS. EphB2 can mediate axonal development by providing retractive cues to assist the axon to reach the target, but EphB2 can also promote dendritic branching to improve learning and memory, which would be particularly beneficial for HAND patients that experience cognitive deficits. We observed a correlation between the upregulation of EphB2 in response to TNF-α and neurite outgrowth, which provides a potential pathway to repair damaged neurons and re-establish lost neuronal connections. Dendritic pruning and neuronal loss has been observed in HAND patients, so this ability to promote repair could prevent, improve, or recover the cognitive deficits experienced by HIV-patients with HAND. TNF-α, although primarily known to induce neurotoxicity, strongly activates the nuclear factor-kappaB (NF-κB) pathway, which can have a very wide range of transcriptional effects. Therefore, our hypothesis is that the TNF-α-induced neurite regrowth occurs through an upregulation EphB2 in an NF-κB-dependent pathway. TNF-α has been well established to induce NF-κB signaling, mostly by promoting the translocation of the NF-κB p65 DNA binding factor to the nucleus for transcriptional regulatory effects. NF-κB can regulate neuronal growth and process development of both dendrites and axons, which would correlate to the neurite regrowth observed following TNF-α upon induced injury. The regulation of EphB2 by NF-κB has not been extensively studied, but EphB2 can be negatively regulated by an NF-κB family member, c-Rel. We analyzed the EphB2 promoter and identified three NF-κB p65 binding sites upstream from the transcriptional start site, which provided insight to our hypothesis. We established that p65 directly binds to and can regulate EphB2 promoter activity in response to TNF-α. Since the dual role of TNF-α can be dependent on the receptor through which the signaling proceeds, either TNF-α receptor 1 (TNFR1) or TNF-α receptor 1 (TNFR2), we investigated if this upregulation of EphB2 is receptor dependent and determined EphB2 is induced primarily through activation of TNFR2. Neurons express both receptors, however, the effects of TNF-α to promote neuroprotection and repair primarily occur through the TNF-α/TNFR2 regulatory axis. Although we have been established the mechanism of TNF-α-induced EphB2 and there is a strong correlation with neurite outgrowth following induced injury, we considered the possibilities to modulate EphB2 in the absence of TNF-α to demonstrate the direct effects of EphB2 expression. Several approaches could be used to mediate EphB2 activation or inhibition in vitro. RNA interfering techniques, such as small interfering RNA (siRNA), are useful, but we were interested in a complete knockout strategy. Since our approach was to assess the effects of EphB2 knockout only on neurite outgrowth following induced injury, a knockout animal model would not be appropriate, as a lack of EphB2 would affect the development of the neurons, unless an inducible knockout model was established. This is a lengthy and elaborate process and, more importantly, would only be available in a non-human model. Other techniques, such as transcription activator like effector nucleases (TALENs), can generate knockout systems that are targeted to specific regions of a gene, but specific binding proteins must be created to recruit the endonucleases to the target. Clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) has emerged as a specific and relatively easy technique to knockout genes of interest and uses short RNA sequences to guide Cas9 endonucleases to target regions to create double stranded breaks in the DNA to silence the gene. Once concern with Cas9 is specificity to target only the desired region of the gene, as off-target effects can occur and may result in unwanted gene silencing. A Cas9 mutant, Cas9 nickase (Cas9n), has been created to have more specificity by requiring two guide RNAs to recruit two Cas9 nickases to generate a double stranded break as they function as nickases to only create a nick in one DNA strand. We developed this strategy to remove exon 1 of the EphB2 gene by using two pairs of Cas9 nickases, with four guide RNAs, to eliminate any chance for off-target effects but retaining the desired outcome of and EphB2 knockout. We validated the system by demonstrating that a knockout of EphB2 increases adhesion and prevents migration in human embryonic kidney 293T (HEK293T) cells. Although this cell model is not a neuronal cell model, the migration assay demonstrates the functional loss of EphB2. We also created an inducible Ephb2 system to overexpress EphB2. Together these provide essential tools to verify the direct involvement of EphB2 in neurite outgrowth. Taken together, our studies characterize a novel mechanism for neurite outgrowth following injury in neurons: TNF-α/TNFR2-induced EphB2 signaling in an NF-κB p65-dependent manner. In addition to the established mechanism, we developed a technique to assess the effects of EphB2 knockout and overexpression in the context of neurite outgrowth: EphB2-targeted-Cas9n and EphB2 inducible construct. This mechanism yields insight into a potential downstream pathway to be utilized to repair damaged regions in the brain and reverse cognitive deficits in neurodegenerative conditions, especially in a chronic inflammatory environment, such as HIV-1 infection. The strategies created provide a valuable toolset to demonstrate the direct effects of modulating EphB2 signaling, not only in neurons for effects on neuronal health and synaptic plasticity, but also in other disease models, such as glioblastoma, in which EphB2 was demonstrated to promote invasion and migration of tumor cells. These observations and the usefulness of the modulatory strategies likely extend to multiple neurodegenerative diseases that demonstrate cognitive deficits that correlate to neuroinflammation. / Biomedical Neuroscience

Total syntheses of the neuroregenerative natural products vinaxanthone and xanthofulvin have been accomplished. The synthetic routes to both molecules utilize a highly regioselective furan Diels-Alder cycloaddition - aromatization sequence to furnish the catechol fragment present in both natural products. The pentasubstituted catechol was elaborated to a vinylogous amide which was used twice in both syntheses, exploiting the pseudosymmetry found in vinaxanthone and xanthofulvin. This approach enabled the dimerization of 5,6-dehydropolivione forming vinaxanthone, lending significant evidence to a non-enzymatically driven formation of vinaxanthone in Nature. The total synthesis of vinaxanthone was accomplished in nine steps, the shortest synthesis to date, and an additional route was devised to access a set of analogs for biological study. The first total synthesis of xanthofulvin was accomplished in 18 steps and the convergent nature of the synthetic plan allows for analog synthesis. The sets of vinaxanthone and xanthofulvin analogs will be used to examine their inhibition of Semaphorin3A, a protein which inhibits neuronal regeneration, and is the biological target for both molecules.

Indiana University-Purdue University Indianapolis (IUPUI) / Traumatic brain injury (TBI) is a critical public health issue in the United States, affecting about 2.8 million people annually. Extensive cell death and neural degeneration directly and diffusively caused by the initial mechanical insult results in a wide range of neurological complications post-trauma. Learning and memory dysfunction is one of the most common complains. Hippocampal neuronal loss, together with other mechanisms, largely contributes to learning and memory impairment as well as other cognitive dysfunctions post-trauma. To date, no FDA-approved drug is available to target cell death or improve learning and memory following TBI. It is of great interest to develop alternative approaches targeting neural repair instead. Neural stem/progenitor cells (NSCs) in the adult hippocampus undergo life-long neurogenesis supporting learning and memory functions, thus hold great promise for post-traumatic neuronal replacement. The previous studies demonstrated that TBI transiently increase NSC proliferation. However, it is debated on whether TBI affects neurogenesis. The mechanism of TBI-enhanced NSC proliferation remains elusive. In the current studies, I have investigated post-traumatic neurogenesis after different injury severities, evaluated integration of post-injury born neurons, illustrated a molecular mechanism mediating TBI-enhanced NSC proliferation, proposed a de novo state of NSCs, and tested effects of a pharmacological approach on spatial learning and memory function recovery. My results demonstrated that post-traumatic neurogenesis is affected by injury severities, partially explained the pre-existing inconsistency among works from different groups. Post-injury born neurons integrate in neural network and receive local and distal inputs. TBI promotes functional recruitment of post-injury born neurons into neural circuits. Mechanistically, mechanistic target of rapamycin (mTOR) pathway is required primarily for TBI-enhanced NSC proliferation; NSCs feature a de novo alert state, in which NSCs are reversibly released from quiescence and primed for proliferation. Furthermore, my data demonstrated a beneficial role of ketamine in improving post-traumatic spatial learning possibly by activating mTOR signal in NSCs and/or promoting neuronal activity of post-injury born neurons. Together, my data support the feasibility of neurogenesis mediated neuronal replacement, provide a target for enhancing post-traumatic NSC proliferation and subsequent neurogenesis, and prove a potential pharmacological approach benefiting post-traumatic functional recovery in learning and memory. / 2021-11-04

No description available.

No description available.

Repair of the adult mammalian spinal cord is prohibited by several extrinsic and intrinsic factors. As the CNS matures, growth-promoting proteins such as integrins are developmentally downregulated resulting in a reduced capacity for axonal outgrowth. Integrins are heterodimeric receptors involved in cell-cell and cell-matrix interactions. Specifically, within mature corticospinal tract (CST) axons, integrins are not transported into the axonal compartment. One integrin heterodimer, α9β1, is of particular interest for its ability to promote neurite outgrowth when bound to a component of the injury-induced milieu, tenascin-C. This project aimed to increase integrin expression within the CNS using induced pluripotent stem cell-derived human neural progenitor cells (iPSC-hNPCs). Using immunocytochemistry and western blotting, endogenous integrin expression within iPSC-hNPCs was determined. In addition, overexpression of α9 integrin was achieved using transfection and lentiviral transduction. The capacity of wild type (WT) and α9-hNPCs to extend neurites on tenascin-C was assessed using neurite outgrowth assays. Results revealed increasing α9 integrin expression in hNPCs significantly promoted neurite outgrowth when cultured on tenascin-C. Interestingly, increasing the concentration of human tenascin-C, resulted in increasingly longer neurites from WT hNPCs suggesting hNPCs could actively upregulate integrin expression. Subsequently, WT and α9-hNPCs were transplanted into layer V of the neonatal rat sensorimotor cortex, which projects to the CST. WT and α9-hNPCs survived up to 8 weeks post-transplantation and produced projections along white matter tracts, including areas of the CST. Additionally, hNPCs retained α9-eYFP protein expression in vivo over time and was localised within axonal projections. These results highlight the capabilities of iPSC-hNPCs to promote integrin expression within the rodent CNS presenting one potential avenue to target neuronal replacement following spinal injury. Future research should focus on assessing the regenerative capacity of WT and α9-hNPCs within an injury model concentrating on the ability of these cells to adapt within an injured environment.

Traumatic brain injury (TBI) is the most common cause of mortality and disability in the younger (&lt;50 years) Swedish population with an incidence rate of 20,000 cases per year. This thesis aims to increase the understanding of brain injury mechanisms, especially in a molecular and cellular context. Bone morphogenetic protein (BMP) signalling was examined in three genetically modified mice (two “loss-of-function”, one “gain-of-function”) exposed to TBI (controlled cortical impact, CCI) with CaMKII used as promoter for Cre-driven recombination in postnatal forebrain neurons. The mice survived, developed normally and did not show any obvious phenotypes except for an upregulation in Mtap2 mRNA in mice with impaired BMP signalling. Reactive Gfap and Timp1 mRNA expression measured using quantitative RT-PCR (qRT-PCR) was reduced in the mice overexpressing BMP signals. The BMP signalling pathway was further studied in cultured PC12 cells with BMP4 and NGF added. Egr3 expression was substantially increased by these growth factors. Blocking Egr or Junb functions reduced neurite outgrowth. TBI-induced mRNA expression changes in 100 selected genes in C57BL/6J mouse neocortex and hippocampus were measured using qRT-PCR at different time points post-injury. Several distinct gene clusters with similar expression patterns were identified. GeneChip analysis (Affymetrix) of the injured mouse neocortex at three days revealed 146 transcripts significantly upregulated, confirming and extending the qRT-PCR results. The findings demonstrate marked increases after injury among chemokine transcripts and activation of many genes involved in inflammation. In conclusion, the present study has revealed transcriptional changes in specific signalling pathways after brain injury. The results may help to identify novel targets for neuroprotective interventions after traumatic brain injury.

Constant neuroregeneration in adult olfactory epithelium maintains olfactory function by basal stem cell proliferation and differentiation to replace lost olfactory sensory neurons (OSNs). Understanding the mechanisms regulating this process could reveal potential therapeutic targets for stimulating adult olfactory neurogenesis under pathological conditions and aging. Ciliary neurotrophic factor (CNTF) in astrocytes promotes forebrain neurogenesis but its function in the olfactory system is unknown. Here, we show in mouse olfactory epithelium that CNTF is expressed in horizontal basal cells, olfactory ensheathing cells (OECs) and a small subpopulation of OSNs. CNTF receptor alpha was expressed in Mash1-positive globose basal cells (GBCs) and OECs. Thus, CNTF may affect GBCs in a paracrine manner. CNTF−/− mice did not display altered GBC proliferation or olfactory function, suggesting that CNTF is not involved in basal olfactory renewal or that they developed compensatory mechanisms. Therefore, we tested the effect of increased CNTF in wild type mice. Intranasal instillation of a focal adhesion kinase (FAK) inhibitor, FAK14, upregulated CNTF expression. FAK14 also promoted GBC proliferation, neuronal differentiation and basal stem cell self-renewal but had no effective in CNTF−/− mice, suggesting that FAK inhibition promotes olfactory neuroregeneration through CNTF, making them potential targets to treat sensorineural anosmia due to OSN loss.