Neural tube defects (NTDs) impact 3000 pregnancies a year in the US and are caused by both genetic and environmental factors. NTDs arise from errors in neural progenitor signaling, migration, proliferation, and differentiation during embryonic development. Spina bifida (SB), a prevalent NTD, can reduce the functioning of neural pathways responsible for pain and motor function in the lower body. A recent study discovered a novel variant of the receptor GPR161 present in screened infants with SB, but absent in all screened infants without. GPR161 is a G protein-coupled receptor localized in the primary cilia known to participate in the regulation of the pathways of key stem cell differentiation ligands, sonic hedgehog (Shh) and Wnt. Our study seeks to investigate the molecular mechanisms which connect the novel GPR161 variant p.Trp202Gly to neural tube defects using an in vitro model of neural stem cell differentiation. GPR161 variant and knock-out (K/O) lines are generated using CRISPR Cas9 technology in induced pluripotent stem cells (iPSCs). iPSCs are then guided through neural differentiation and harvested for analysis at multiple key stages of neural progenitor development. Markers of neural differentiation, SB, and downstream GPR161 factors are analyzed using western blot, RT-qPCR, immunostaining, and RNAseq. We expect to see a change in Shh activity in the variant line compared to the WT.

Chronic respiratory infections and diseases are the third leading cause of death globally. The Frevert lab studies the protein versican (Vcan), an extracellular matrix proteoglycan, whose expression is highly upregulated during lung injury and inflammation. However, it is unclear whether this upregulation is an anti-inflammatory or a pro-inflammatory response. We are testing the hypothesis that the cellular source of Vcan determines its inflammatory actions; versican from myeloid cells is anti-inflammatory but versican from stromal cells is pro-inflammatory. Vcan deletion in our cells of interest can test this hypothesis. To do this we have generated Vcan^fl/fl genetically engineered mice, which allow for conditional removal of functional versican with Cre Recombinase. Cre excises versican’s exon 4, recombining exons 3-5 and creating a premature stop codon. This produces a truncated non-functional form of versican. The goal of this research project is the development of cell-specific protocols for in vitro deletion of versican in both myeloid cells (bone marrow-derived macrophages) and stromal cells (lung explant fibroblasts). These protocols will allow for further evaluation of versican’s role in the inflammatory response when different cell types are confronted by a bacterial or viral agonist. So far, I’ve been investigating the dose response and time course for exposure of macrophages to Cre to optimize its efficiency and have been able to demonstrate by qPCR that intact versican decreases and non-functional versican increases. The inflammatory response is quantified through qPCR analysis of the fold increase of Ifn-b compared to the house-keeping gene MRPL32. Ifn-b is a cytokine released by the innate immune system in response to viral pathogens. Next, I will investigate the conditions necessary for efficient Cre deletion of Vcan in fibroblasts. These experiments allow us to investigate the effects of Vcan made by different cell types furthering our understanding Vcan’s function during injury and inflammation.

Sleep deprivation (SD) is a major health concern in developed countries, especially in the elderly, and is associated with physiological disturbances including cognitive impairment, increased risk of dementia, and leads to the acceleration of neurodegenerative disorders such as Alzheimer’s disease. A logical question is whether cognitive impairment associated with SD can be prevented. To address this question, 22-month-old C57BL/6 mice were sleep deprived during the middle of their sleep cycle for four hours each day for two days. Three days before the SD procedure, treatment was started with the anti-aging peptide glycyl-L-histidyl-L-lysine (GHK) 15 mg/kg per day for five days through the two-day SD procedure. GHK has wound healing and anti-inflammatory properties and readily passes through the blood-brain barrier. Immediately following the last day of SD and GHK treatment, mice were tested for learning impairment using a spatial learning activity which requires them to find the escape hole. Tissues were then collected for neuropathology assessment. Sleep deprived mice treated with GHK consistently found the escape hole more quickly than sleep deprived mice treated with saline suggesting that GHK can prevent the learning impairment associated with short term sleep deprivation. This preliminary observation provides the rationale to investigate the neuro-molecular and neuropathological pathways targeted by GHK including inflammation, oxidative stress and vascular dysfunction.

Astrocytes are characteristic star-shaped glial cells, and they are the largest and most numerous nonneuronal cells in the brain. They are involved in cell signaling, providing nutrients to neurons, removing toxins, repair and anti-inflammation response in the brain. However, relatively little attention has been paid to this cell type compared to neurons in neurodegenerative conditions, such as age-related cognitive impairment and Alzheimer’s disease. The basic question is: what functional changes occur in astrocytes with increasing age that could relate to vulnerability to neurodegeneration. C57BL/6 mice in age cohorts of 8 months, 16 months, 24 months, and 32 months were tested in a radial water tread maze, a well-standardized measure of contextual learning and memory in the mice. Brain tissues were then collected and parasagitally sectioned and stained with GFAP (Glial Fibrillary Acidic Protein), an immuno-reactive marker for astrocytes. In general, 8-month old mice showed very little cognitive impariment while several 16-month old mice showed mild cognitive impairment, but 24 and 32-month old mice showed moderate to severe cognitive impairment. Concurrent with this increase in cognitive impairment was an increase in ImageJ pixel intensity of GFAP immunohistochemistry staining of astrocytes with increasing age. This preliminary observation suggests that astrocytes are predominantly present with increasing age and decreasing cognitive ability and provides the rationale for further investigations into what role these nonneuronal cells are playing in brain health.

Senescence is a gradual deterioration of functional characteristics. At the cellular level, it is defined as a loss of the ability to divide and carryout normal cellular activities that contribute to the health and well being of a cell. In fact, senescent cells secrete inflammatory cytokines that contribute to aging. Therefore, preventing senescence could have an impact on preventing aging. To address this issue, 21-month-old C57BL/6 mice were fed standard rodent chow containing three well-validated anti-aging drugs, rapamycin, acarbose, and phenylbutyrate for 3 months. Each drug targets different but overlapping aging processes. At the end of 3 months mice were tested for cognition and physiological performance and tissues collected for assessing senescence by reverse transcriptase polymerase chain reaction using well-accepted senescence markers including p16. Preliminary observations suggest that the drug cocktail in just 3 months delayed aging by improving cognitive and physiological performance tasks. Preliminary data from liver samples tested by reverse transcriptase polymerase chain reaction showed that mice treated with the drug cocktail had less P16 expression than mice fed a diet without the drug cocktail. More work is needed to sort out how each drug is contributing to this anti-senescence effect, but the preliminary observation suggests preventing or delaying senescence may be an informative target for investigations into delaying or preventing aging.

Diets high in fat and sugar have increasingly adverse effects with increasing age because of generally decreasing activity and energy needs. This nutritional overload results in an increase in body fat mass and an increase in risk for metabolic and cardiovascular disease and possibly other chronic diseases such as cancer. However, the role of fat mass in age-related pathology and survival over an extended period of time is controversial. To address this issue, C57BL/6 mice, 18 months of age, were started on a high caloric (HC) diet consisting of balanced protein, lard, and sucrose. A second cohort was maintained on a standard rodent caloric (RC) diet consisting of balanced protein, wheat, corn, and soybean oil. Fat mass was measured by quantitative magnetic resonance imaging (QMRI) monthly for 10 months (28 months of age) at which time mice were evaluated for physiological performance, and tissues collected for geropathology. Both diet groups started with an average 5 percent fat mass. Fat mass increased to 15 percent in the HC diet group over the next 3-5 months, then gradually decreased to 9 percent after 10 months on the diet. Fat mass gradually decreased to 3 percent in the RC diet group over 10 months. Mice fed the RC diet had increased paw grip strength and were able to stay on a rotating rod longer than mice fed the HC diet, but there was no difference in survival between the two diet cohorts over the 10-month trial period. These preliminary observations suggest that percent body fat mass generated by a diet high in animal fat and table sugar may be associated with unhealthy aging, but not survival. This provides rationale for subsequent investigations into the pathological consequences of high caloric diets with increasing age.