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Cell Culturing on 3D Printed LCEs
The purpose of this study is to culture cells on 3D printed structures known as liquid crystal elastomers (LCEs) using templates from microscope scanned tissue specimens. LCEs are composed of a long crosslink of polymers that have a porous property to them that will aid in growth and proliferation of the glial cells once they are seeded into the material. The process by which this is possible is photo curing resin of different types by using multiple formulations of elastomers that contain varying polymerizers. Two main forms of printing are used to accomplish this; an SLA UV curing printer and the use of a custom designed microscope setup. The SLA UV curing printer cures on a layer-by-layer basis at ~30 micron resolution. While the custom designed setup allows for the use of a laser scanning microscope which is capable of providing finer resolution for the LCEs. So far, through the use of the SLA UV curing printer, our lab has been able to print structures of tissue that contain all the characteristics that would make the growth of glial cells a feasible task. The structures were printed using modified materials which contain photo-initiators that enable the material to undergo physical property changes when they react with the UV light from the printer. More specifically, it was a compound by the name of 6arms-alpha (modified), which contains 10% of liquid crystal and has a high viscosity. The importance of this research is to provide a way to study neurons in arrangements that emulate real tissue, which could have a significant impact in the future of bioimplants.
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Impact of the Methyl Donor Betaine on Repairing Mitochondria in Neurodegenerative Disease
In Alzheimer’s disease (AD) and multiple sclerosis (MS) histone methylation is severely reduced. This hypomethylation leads to aberrant gene expression that brings about disease-causing plaque and lesion buildup within the central nervous system. It is known that this aberrant gene expression affects mitochondria in these diseases. I hypothesized that introducing methyl donors could prevent hypomethylation of histone H3, as well as dysfunction of mitochondria in mouse models of AD and MS. I tested the effects of the methyl donor betaine (also known as trimethylglycine). We administered betaine in drinking water to the APP/PS1 mouse model of AD, and the Cuprizone mouse model of MS for four weeks. The aim of this study was to measure the protective effects of the methyl donor betaine on mitochondrial complexes downregulated in neurodegenerative diseases. Protein was isolated from the brains of mice with and without betaine. The levels of different mitochondrial complex genes in the presence and absence of betaine were measured using Western blotting, JC1 staining, immunohistochemistry (IHC), and densitometry. This research has important implications for developing new therapies to treat AD and MS.
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Actigraphy-Based Circadian Measures and Cerebrospinal Fluid Biomarkers of Neurodegeneration in Alzheimer’s Disease with Mild Cognitive Impairment
Although recent data implicates sleep and circadian disruption to neurodegeneration in Alzheimer’s Disease (AD), the association of objective circadian biomarkers and neurodegeneration remains understudied. We hypothesize that actigraphy-based circadian measures are associated with cerebrospinal fluid (CSF) biomarkers of neurodegeneration in those mild cognitive impairment due to AD (MCI-AD).Eighteen patients with CSF biomarker-confirmed MCI-AD underwent actigraphy monitoring generating the following circadian measures: amplitude, F-ratio and mesor and morning collection of CSF biomarkers of neurodegeneration (Aβ42,t-tau,p-tau). Linear models were used to evaluate the association of circadian and CSF measures; logarithmic transformations were performed on neurodegenerative markers for greater normality. Analysis was performed using SAS software. A significance level of 0.05 was assumed for all tests. Eighteen MCI-AD patients who were 68± 6.2 years, 44% female, with median AHI=12 and underwent actigraphy monitoring for 8.2+/-3.2 days were included. There was no significant association of circadian measures and Aβ42 nor with mesor and neurodegeneration biomarkers. Amplitude was associated with both p-tau and t-tau, such that each 10 unit increase in amplitude resulted in a predicted increase in p-tau of 8% (95% CI: 1%-15%, p=0.018) and an increase of 13% (3%-23%; p=0.01) in t-tau. F-ratio was positively associated with p-tau and t-tau; each 1000 unit increase in F-ratio resulted in a predicted 12% (4%-22%; p=0.007) increase in P-tau and 20% (6%-35%; p=0.005) increase in t-tau. Associations of these circadian measures and CSF levels of p-tau and t-tau remained statistically significant after adjustment for age and sex. Among patients with symptomatic MCI stages of AD, objective measures of circadian rhythm disruption are associated with CSF-based biomarkers of neurodegeneration even after consideration of age and sex.
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Treating Neuropathology with the Novel Hormone Irisin
Alzheimer’s disease is characterized by neuroinflammation and neural accumulation of pathological tau. One candidate for treatment is a recently discovered hormone irisin, which is a cleaved product from the fibronectin type III domain-containing protein 5 (FNDC5) transmembrane protein. We have preliminary data showing that weekly injections of recombinant irisin decreased neuroinflammation and tau pathology in 4-month old female htau mice that express a mutant, humanized gene associated with late-onset Alzheimer’s disease. In the current study, we determined how changes in treatment frequency and age affected neuropathology, inflammation, and FNDC5 levels in htau mice and their wildtype littermates that lack any form of tau.
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Impact of Physical Activity on CNS Mitochondrial Metabolism School of Biomedical Sciences, Kent State University Dept. of Biological Sciences, Kent State University Brain Health Research Institute, Kent State University
Multiple sclerosis (MS) is a complex neurodegenerative disease of the central nervous system (CNS) and dysfunctional mitochondria in neurons have been suggested to play a role. Hemoglobin is present in cortical neurons and levels are altered in MS cortex. The role of hemoglobin is unclear, however, hemoglobin β (Hbb) subunit levels are increased in mitochondrial fraction of MS cortex suggesting a potential role of hemoglobin in mitochondrial respiration. In fact, Hbb is tightly bound to chromatin and mediates a signaling mechanism that supports neuronal energetics involving the trimethylation of histone H3 on lysine 4 (H3K4me3), a histone mark that activates transcription of mitochondrial genes and increases mitochondrial respiratory capacity. We are excited by the potential influence that exercise may have on Hbb expression in the CNS and its impact on neuronal energetics. Thus, we compared exercise and sedentary lifestyles on neuronal Hbb expression and mitochondrial metabolism using Sprague Dawley rats. Rats aging 6-8 weeks-old were provided running wheels for a 7 week period. The Hbb expression was shown to be increased in exercised rats compared to sedentary rats. We also observed that increased Hbb expression correlated with an increase in basal neuronal mitochondrial respiration and increased mitochondrial respiration was linked to elevated levels of the neuronal mitochondrial metabolite N-acetylaspartate (NAA). Understanding regulatory mechanisms that impact Hbb expression and how Hbb supports neuronal mitochondria could lead to new therapeutic approaches (including exercise) to treat neurodegenerative diseases including MS, Alzheimer’s, and Parkinson’s where dysfunctional mitochondria contribute to disease activity.
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Ferret Odor Induces Novel Gene Pathways in the Ventromedial Hypothalamus
Nearly 40% of Americans are obese and this can be countered by increasing calories burned. Muscle thermogenesis is a possible untapped source for increasing energy expenditure. Elevating thermogenesis increases energy expenditure and is linked to weight loss. Our research program has shown that exposing rats to predator odor (PO) induces a rapid and robust increase in muscle thermogenesis that peaks by 30 min and settles to near baseline after 4 hours. We hypothesize that this is mediated in the brain by the ventromedial hypothalamus (VMH) because of the importance of the VMH in regulating body weight, muscle metabolism, sympathetic nervous system activity, and the behavioral response to predator threat. We investigated possible PO-induced changes in the VMH by exposing rats to predator odor for 30 minutes to 4 hours. Tissue samples were taken from the VMH, and mRNA expression was measured using qPCR, showing an increase in Sirt 1 and Bdnf, but not SF1, after 4 hours of PO exposure. RNA-sequencing analyses identified 164 differentially expressed genes (DEGs), 65 enriched gene ontology terms, and 28 genome pathways revealing themes of immune response, oxidative stress, and synaptic plasticity. This allowed us to conclude that PO exposure causes upregulation of novel genes pathways.
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Effects of Early Life Neglect on Adolescent Cocaine Use and Expression of Fibroblast Growth Factor-2 in the Brain
Early-life adversity (ELA) increases the risk for development of psychopathologies in adolescence and adulthood. Childhood neglect is a potent form of ELA and can be modeled through neonatal maternal separation. Maternal separation has been shown to alter cognition, learning and memory later in life. Here, we examined the contribution of maternal separation to the development of substance use disorder in adolescence, and whether changes in regional protein expression in the brain persist into adulthood. First, male and female rats exposed to maternal separation were compared to controls for changes in localized expression of the developmental growth factor, fibroblast growth factor-2 (FGF2), in several brain regions using immunohistochemistry. FGF2 is known to be upregulated by cocaine experience, is protective against fear over-expression and is a candidate biomarker for vulnerability and resilience to development of comorbid psychopathologies. Next, we examined whether maternal separation impacted sensitivity in adolescence to cocaine using the conditioned place preference paradigm, in which one of two chambers is associated with cocaine. Finally, we determined whether maternal separation altered regional FGF2 expression in the brain during adulthood, and if cocaine experience in adolescence had an impact. We are currently processing the brain tissue to examine regional FGF2 expression and have completed the conditioned place preference protocol. Our results will determine whether FGF2 is a potential risk or resilience factor for the development of psychopathologies and will pave the way for future studies examining comorbid addiction and fear disorders.
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Mitochondrial Dynamics in Neurodegenerative Mouse Models
Neurodegenerative diseases are becoming more common in today’s world. Two of the more prominent diseases that fall into this category are multiple sclerosis (MS) and Alzheimer’s disease (AD). A hallmark for both of these diseases is mitochondrial dysfunction. Mitochondrial dysfunction causes an increase in the number of damaged mitochondria, which is linked to a decrease in the energy supply for the cell. Mitophagy is a mechanism known for eliminating damaged mitochondria and is impaired in neurodegenerative diseases. This causes an accumulation of damaged mitochondria in the cell leading to loss of function. We have also shown that a dietary factor known as betaine, is able to restore energy production within the dysfunctional mitochondria. In the present study, we are investigating the role of betaine in both AD and cuprizone mouse models, hypothesizing that betaine will restore energy production by eliminating damaged mitochondria using mitophagy.
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The Role of the Locus Coeruleus on Regulating Sickness-like BehaviorsElevated peripheral cytokines result in sickness and depression-like behaviors by signaling through the vagus nerve. Stimulation of the vagus nerve activates noradrenergic neurons in the nucleus of the solitary tract (NTS) and release of norepinephrine (NE) at projection sites that regulate behavior. Previous studies indicate that neurons in the locus coeruleus (LC) inhibit neurons in the NTS. This led to the hypothesis that LC neurons may act as a brake to inhibit sickness behaviors. To test this hypothesis, rats underwent baseline behavioral testing before being injected with either saline or DSP4, a neurotoxin that lesions LC neurons. Three weeks later animals underwent further behavioral testing before all animals received live E.coli injections and sickness behaviors were recorded. We expected lesioning the LC would result in greater sickness behaviors following E.coli by removing the inhibitory regulation of NTS projection neurons; however, lesioned animals had similar body weights, food intake, and sucrose intake compared to controls following E.coli challenge. Interestingly, DSP4 treated rats showed greater anxiety-like behaviors in an open field test three weeks following DPS4 and less sickness behaviors in an open field following E.coli compared to controls. The results did not support our initial hypothesis, but do support a role for LC neurons in regulating sickness and/or depression/anxiety-like behaviors. Future studies will look more into the role the LC plays in regulating behaviors and the interactions between NTS, LC, and prefrontal cortex (an area important for emotional and motivated behaviors). |