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MicroRNAs (miRNAs) are short, non-coding RNA sequences that are generated from longer primary transcripts (pri-miRNA). These pri-miRNAs are processed by the endonuclease Drosha into a hairpin secondary structure (pre-miRNA), exported from the nucleus and cleaved by the enzyme Dicer to form a duplex RNA molecule. This miRNA:miRNA* duplex is subsequently further processed to form a single-stranded, mature miRNA. miRNAs have been extensively characterized and are known to play important roles in various physiologic and pathologic pathways. One hallmark of miRNAs function is their ability to modulate the downstream activities of protein-coding genes, as well as various other aspects of gene expression, by acting as post-transcriptional repressors of their messengerRNA (mRNA) targets. miR-184 is a highly conserved miRNA gene expressed in the Drosophila nervous system throughout development; and has been shown to target key regulators of differentiation, proliferation and apoptosis. Here we identify a novel role for miR-184 in regulating the development and maintenance of the Drosophila melanogaster post-embryonic nervous system. We present evidence which suggest miR-184 targets (i) paralytic (para), a voltage-gated sodium channel, shown to control neuronal excitability; and (ii) tramtrack69 (ttk69), a transcription factor known to regulate glial cell number and fate determination during embryonic development. In the absence of miR-184, homozygous loss-of-function mutant adult flies demonstrate hyperactive episodes in response to mechanical shock, indicative of increased susceptibility to seizures. Homozygous loss-of-function mutants also exhibit shortened lifespan, as well as reduced group longevity. Additionally, miR-184 deficient mutant larvae exhibit abnormal development of glia and glial progenitors; while expression of miR-184 exclusively in glia - reversed polarity- (repo) expressing cells - up-regulates development of glial cells. Phenotypes of the adult loss-of-function mutant are suppressed by genetic loss of para function; while larval phenotypes are rescued by reducing the genetic dosage of ttk69. These data imply that miR-184 functions to control post-embryonic gliogenesis, as well as in maintaining neuronal excitability and integrity of the Drosophila aging brain. / Biology

The human brain is a complex organ that is responsible for regulating all the physiological processes in the body, ranging from memory to movement. As humans age, the brain goes through a variety of changes including a reduction in glymphatic waste clearance and increase in glial reactivity. Two neurological conditions that affect individuals over the age of 65 include glioblastoma (GBM) and Alzheimer's disease (AD). Interestingly, patients with GBM do not present with AD and vice versa. Both conditions are characterized by a disruption in interstitial fluid flow (IFF) and an increase in neuroinflammation. Throughout the following dissertation, we examined the role of IFF in AD and GBM progression using a three-sided approach (in vivo, in vitro, and in silico). Increased IFF underlies glioma invasion into the surrounding tumor microenvironment (TME) in GBM. We used a 3D hydrogel model of the GBM TME to examine potential pathways by which astrocytes and microglia contribute to glioma invasion. A reduction in IFF contributes to accumulation of the toxic protein amyloid beta (Aβ) in AD. We sought to create a novel, patient-inspired model of the AD hippocampus for examination of the relationship between IFF and Aβ clearance. Human AD and unaffected control hippocampal brain samples were stained for markers of neurons, astrocytes, microglia and Aβ. The percentage of each cell population in the CA1 region of the hippocampus was calculated. We also analyzed the amount and characteristics of the Aβ aggregates present in this hippocampal region. Pearson correlation analysis was completed to assess the relationships between the various cell populations, Aβ load, and patient descriptors. The cell ratios gleaned from the patient samples were incorporated into a novel, 3D hydrogel model of the AD hippocampus. This model features a hydrogel mixture like the native brain extracellular matrix (ECM) and allows for the application of IFF and Aβ. To our knowledge, we are the first group to create a patient-specific triculture model of the AD hippocampus, which is the main site of Aβ aggregation in the AD brain. We used this model to examine the relationship between IFF-mediated Aβ clearance and glial reactivity. The last aim of this dissertation was to create a computational model for examining Aβ binding within the ECM and the effects of IFF on Aβ clearance. In vitro experiments were conducted to generate 3D renderings of glial cells and to determine relevant parameters for our model. Throughout this work, we discuss the relationship between disruption in IFF and glial reactivity in the context of GBM and AD. / Doctor of Philosophy / The human brain is a complex organ that is responsible for regulating all the physiological processes in the body, ranging from memory to movement. As humans age, the brain goes through a variety of changes including a reduction in brain waste removal and an increase in inflammation. Two neurological conditions that affect individuals over the age of 65 include glioblastoma (GBM) and Alzheimer's disease (AD). Interestingly, patients with GBM do not present with AD and vice versa. Both conditions are characterized by a disruption in brain interstitial fluid flow (IFF) and an increase in neuroinflammation. Throughout the following dissertation, we examined the role of IFF in AD and GBM progression using a three-sided approach including analysis of mouse and human tissues, engineered cell models, and computational methods. Specific interactions between brain cell types and their relationships with glioma invasion were examined using a 3D cell model that mimics the brain. Through the work presented here, we also sought to create a novel cell model of the hippocampus region located in the AD brain. We quantified the various cell types in the hippocampus of AD patient samples and incorporated this information into our hydrogel model. The resulting model features three brain cell types (astrocytes, microglia, and neurons) that are added at patient relevant ratios, a matrix that mimics the native brain scaffold, and allows for the application of IFF. In the AD brain there is a reduction in brain waste removal that leads to accumulation of the toxic protein amyloid beta (Aβ). We were successfully able to incorporate this protein within our model so we could assess the relationship between IFF and Aβ removal from the brain. We further studied this relationship using a new computational model of Aβ accumulation in the brain. Throughout this work, we discuss the connection between disrupted IFF and neuroinflammation in the context of GBM and AD.

Yes / Background: Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson’s disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results: Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions: The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

Yes / Background: Platelet rich plasma (PRP), processed from autologous peripheral blood, is used to treat androgenetic alopecia (AGA). Objective: To determine the efficacy of PRP for hair growth promotion in AGA patients in a randomized, blinded, placebo controlled, pilot clinical trial (NCT02074943). Methods: The efficacy of an 8 week, 5 session, PRP treatment course was determined by measuring hair density and hair caliber changes in 10 AGA affected patients. For each PRP sample, the concentrations of selected growth factors were determined using a multiplex assay system. The clinical results were then correlated to the growth factor concentrations in PRP. Results: At 16 weeks, 8 weeks after the last PRP injection, treated areas exhibited increased mean hair density (+12.76%) over baseline compared to placebo (+0.99%). Mean hair caliber decreased in both treated and placebo regions (-16.22% and -19.46% respectively). Serial analysis of PRP significant variability in concentrations between patients. Overall, there was a positive correlation between GDNF concentration and hair density (p= 0.004). Trends, though not statistically significant, were also observed for FGF2 and VEGF. Limitations: Small sample size and lack of comparative cohorts receiving protocol variations limit confidence in the study data. Conclusions: This small pilot clinical trial suggests PRP treatment may be beneficial for AGA. However, the variable hair growth responses between patients indicate there is a significant opportunity to improve PRP therapy protocols for hair growth promotion. The variability in growth factor concentration in PRP suggests standardization of growth factors post-processing might improve hair growth responses. / RepliCel Life Sciences Inc. (Canada)