Cheryl Lea Wellington
Relevant Degree Programs
Relationship of biomechanics to pathology in TBI animal models Translational biomarkers for TBI across clinical and animal specimens ApoE modulators Cerebrovascular contributions to Alzheimer's Disease pathogenesis Animal Magnetic Resonance Imaging
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Traumatic brain injury (TBI) is an important public health issue worldwide. It is strongly linked to neurodegenerative conditions such as Alzheimer’s Disease (AD) and Chronic Traumatic Encephalopathy. Despite numerous promising pre-clinical studies, there is no effective clinical treatment for TBI, indicating inefficiency in translation of scientific research from bench to bedside.This thesis thus attempts to address the issue through two approaches. Firstly, we reviewed the commonly-used pre-clinical TBI models, and found that many lack thorough biomechanical considerations. We thus developed a new mouse TBI model, Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA). CHIMERA reproducibly induces clinically-relevant TBI, both in terms of biomechanics and neuropathology. CHIMERA mild TBI (mTBI) induces behavioural (e.g. neurological, motor, and cognitive), histological and biochemical changes (e.g. diffuse axonal injury, white matter microgliosis, and brain cytokine induction). We further demonstrated that CHIMERA mTBI outcomes are scalable by varying the mechanical inputs. These observations demonstrate that CHIMERA is a novel and valuable platform for TBI research.Next, we induced CHIMERA mTBI in APP/PS1 mice, a transgenic model of AD amyloidosis, and characterised both acute and long-term consequences. Here we included two age groups of animals, as TBI occur in both the young and the old populations. In the acute phase, mTBI led to subtle and transient age-dependent changes in A-beta deposits. Age-at-injury and genotype showed complex interactions in determining microglial and cytokine outcomes, such that neuroinflammation was increased in old wildtype mice and young APP/PS1 mice. Age-at-injury also markedly affected neurofilament response, as neurofilament-positive axonal bulbs and plasma neurofilament-light levels were elevated in young mice, but not old mice, of both genotypes. In the chronic phase, mTBI led to prolonged white matter microgliosis and axonal injury up to 8-mo post-injury. MTBI also intensified long-term fear memory in APP/PS1 mice, reminiscent of post-traumatic stress disorder phenotypes.In summary, we have developed a reproducible and clinically-relevant TBI model. We showed that genetic predisposition to AD and age-at-injury are both significant modifiers of acute and long-term mTBI outcomes. These findings may provide insights for future attempts in understanding the mechanistic pathways of TBI pathogenesis.
Traumatic brain injury (TBI) is a “silent epidemic” that currently lacks any effective treatment. While a major health care problem in itself, TBI also increases Alzheimer’s disease (AD) risk and leads to the deposition of neurofibrillary tangles and amyloid deposits similar to those found in AD. Agonists of Liver X receptors (LXRs), which regulate the expression of many genes involved in lipid homeostasis and inflammation, improve cognition and reduce neuropathology in AD mice. One pathway by which LXR agonists exert their beneficial effects is through ATP-binding cassette transporter A1-mediated lipid transport onto apolipoprotein E (apoE). In the first part of this thesis, I show that a short-term treatment with synthetic LXR agonist GW3965 improves post-injury outcomes in mice subjected to closed-head, mild, repetitive weight drop TBI (mrTBI). My results suggest that both apoE-dependent and apoE-independent pathways contribute to the ability of GW3965 to promote recovery from mrTBI. While many drugs have shown promising outcomes in preclinical TBI models, clinical drug trials for TBI so far have failed, suggesting that the translational potential of TBI models may require further improvement. As most human TBIs result from impact to an intact skull, closed head injury (CHI) rodent models are highly relevant. Traditional CHI models like weight drop however suffer from large experimental variability that may be due to poor control over biomechanical inputs. To address this caveat we developed a novel CHI model called CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) that fully integrates biomechanical, behavioral, and neuropathological analyses. CHIMERA is distinct from existing neurotrauma model systems in that it uses a completely non-surgical procedure to precisely deliver impacts of prescribed dynamic characteristics to a closed skull while enabling kinematic analysis of unconstrained head movement. Here I show that repeated TBI in mice using CHIMERA mimics many features of the human TBI including neurological, motor, and cognitive deficits along with persistent neuroinflammation and diffuse axonal injury, and increased endogenous tau phosphorylation up to 14 days with a reliable biomechanical response of the head. This makes CHIMERA well suited to investigate the pathophysiology of TBI and for drug development programs.
Patients with Alzheimer’s Disease (AD) exhibit substantial cerebrovascular damage, including the accumulation of β-amyloid (Aβ) peptides within the vessel wall. Mid-life vascular risk factors increase the risk of AD potentially via the loss of beneficial or gain of toxic functions in circulating high density lipoprotein (HDL). Low plasma levels of apolipoprotein A-I (apoA-I), the primary protein component of HDL, increase AD risk and correlate with cognitive decline, and preliminary preclinical evidence supports a role of apoA-I in mediating removal of cerebrovascular Aβ, suppressing neuroinflammation, and enhancing cognitive function. Our strategy was to perturb peripheral and central nervous system (CNS) apoA-I levels through genetic modification of proteins known to regulate apoA-I metabolism and via indirect and direct pharmacological manipulation of apoA-I to delineate its CNS transport, regulation and therapeutic potential in AD. Loss of ATP binding cassette transporter A1 (ABCA1), which effluxes cholesterol onto lipid-poor apoA-I to generate immature pre-β-HDL, lead to significant parallel decreases of circulating and CNS apoA-I, while stimulation of ABCA1 activity with an Liver-X-Receptor (LXR) agonist substantially increased apoA-I levels selectively in the CNS, solubilized Aβ and improved cognitive function in AD mice. Although apoA-I was increased independent of ABCA1, ABCA1 was required to observe LXR-mediated cognitive benefits, suggesting lipidation of apolipoproteins is a critical regulator of their function. Pre-β-HDL appear to be the more biologically relevant species regarding CNS health, as loss of lecithin-cholesterol acetyl transferase (LCAT), which esterifies free cholesterol to generate mature α-HDL, does not impact AD pathology in vivo. Intravenously injected human apoA-I gains access to the CNS predominantly via the blood cerebrospinal fluid barrier, where it is bound, internalized, and transported by the epithelial cells of the choroid plexus in a specific receptor mediated fashion. Weekly injection of reconstituted HDL, formulated to enrich the pre-β pool, into symptomatic AD mice transiently decreased plasma Aβ levels but was unable to modulate brain Aβ, neuroinflammation, or endothelial activation in the experimental paradigm used. Collectively, these data identified ABCA1 generated apoA-I pre-β-HDL species as a key population of HDL subspecies for modulating AD pathology in vivo.
Lipid transport in the brain is coordinated by glia-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. ApoE plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). ATP-binding cassette transporter A1 (ABCA1) effluxes cholesterol and phospholipids to apolipoprotein acceptors including apoE. ABCA1 is a key regulator of apoE levels and lipidation in the brain and deficiency of ABCA1 increases amyloid burden in AD mouse models. Translating these findings to potential therapies for AD will require a more thorough understanding of the biochemical nature of nascent apoE particles generated from glia and of lipoprotein remodeling in the CNS in general. In this thesis, I show that apoE is secreted from wild-type primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Glia lacking ABCA1 secrete only one species of small particles (~8.1nm), which are poorly lipidated, but can accept lipids to form the full repertoire of wild-type apoE particles. Inhibition of apoE receptor function blocks appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor significantly reduces the level of the 8.1 nm particles, suggesting that apoE is preferentially recycled through LDLR. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways.Modulating the expression, secretion or function of apoE may provide potential therapeutic approaches to protect the brain from chronic and acute damage. This thesis shows that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens have negligible effects. Intriguingly, lynestrenol also increases expression of ABCA1 in human astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor (PR) inhibitor RU486 attenuates the effect of progestins on apoE expression in astrocytoma but has no effect on ABCA1 expression in all glial cell models tested, suggesting that PR may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through LXR-dependent and PR-dependent pathways.
Master's Student Supervision (2010 - 2018)
Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. One of the hallmarks of AD is the accumulation of amyloid plaques in the brain. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. The APOE gene is the most validated genetic risk factor for late onset AD, and has well-established associations with amyloid deposition and clearance from the brain. We and others have shown that lipidation of apoE can assist amyloid clearance, raising interest in augmenting apoE function as a proposed therapeutic strategy for AD. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. A subset of AXL receptor tyrosine kinase inhibitors, which we term AXL modulators were identified as positive hits. The objective of this thesis is to dissect the mechanism of action (MoA) by which AXL modulators upregulate apoE expression. We initially understood their dependency on AXL by treating AXL-/- CCF-STTG1 cells generated using CRISPR-Cas9 method with the lead compound. We then determined if Liver X Receptor (LXR) activity was required utilizing LXR knock-out (KO) mouse embryonic fibroblasts (MEF) cells. Immunoblotting analysis of AXL protein indicated the ability of AXL modulators to promote AXL receptor cleavage and stabilize the intracellular domain (ICD). To investigate the role of AXL-ICD in apoE homeostasis, various Axl variants, including WT AXL, kinase-dead AXL mutant, Axl-ICD, and AXL N-terminal fragment were stably reconstituted in AXL-/- CCF-STTG1 cells. ApoE baseline expression was significantly upregulated only upon reconstitution of ICD-containing AXL variants. In summary, AXL protein plays a significant role in apoE homeostasis through its intracellular domain.
Alzheimer‘s Disease (AD) is a progressive neurodegenerative disease that affects millions ofpeople world-wide. It is characterized by amyloid plaques and neurofibrillary tangles in thebrain. Many AD patients also show loss of cerebrovascular integrity, which is thought to lead todecreased capillary flow, neuronal injury and impaired clearance of amyloid beta. Sinceapolipoprotein E (apoE) and high density lipoprotein (HDL) show many beneficial effects in thevasculature in the body, we aimed to test the effects of these lipoproteins on primary humanperivascular cells in the brain. We found that pharmacologically increasing apoE levels withGW3965 in a scratch-wound assay was not associated with changes in pericyte migration.Interestingly, we found that Axl inhibitor A1 slowed pericyte migration without showingchanges in secreted apoE levels, suggesting an apoE-independent pathway for pericytemigration. We also tested whether HDL can attenuate the CypA-NFκB-MMP9 inflammatorypathway associated with apoE4 pericytes, but failed to observe the activation of thisinflammatory pathway in our pericytes. Lastly, we found that when macrophages were treatedwith HDL, Aβ phagocytosis was not changed. Moreover, there were donor differences in theinflammatory response of macrophages to Aβ, making consistent observations difficult. Takentogether, we did not show beneficial effects of lipoproteins on perivascular cell function in thecontext of AD.