We are researching adeno-associated viral (AAV) vector based gene therapy for the rare genetic muscle disorder GNE myopathy (GNEM). This disease manifests as progressive skeletal muscle atrophy leading to restricted ambulatory ability and loss of fine motor skills. GNEM is caused by defects in the GNE gene, negatively impacting the sialic acid biosynthesis pathway. The sialylation of muscle fibers does not occur without this pathway, causing a loss of muscle function due to sialic acid deficiency. AAV gene therapy can be used to alternatively express the GNE gene by directly targeting muscle or utilizing liver expression. Sialylated glycoproteins secreted by the liver can be taken up by muscle and the sialic acid recycled. Our lab plans to advance GNEM AAV gene therapy by combining liver- and muscle-specific promoters to reduce the dose of AAV required for sialylation of muscle fibers. We have cloned six tandem promoters and three control promoters that will be tested in human liver cells (HepG2) and murine myoblast cells (C2C12). The tandem promoters combine different regions of liver and muscle promoters. A double (firefly and renilla) luciferase mechanism is used to indicate promoter strength, where the amount of luminescence from firefly luciferase is measured. Our lab is currently recording the promoter strength and level of transfection in the cells. The ratio between these expressions indicate the overall strength of the promoter. We are also cloning these tandem promoters into AAV vectors with the intent of testing the vectors in mice. The aim of our research is to produce a promoter resulting in the expression of the GNE gene in both muscle and liver cells, increasing sialic acid production to aid patients affected by GNEM using a minimal dosage.

The Davis lab is focused on understanding the pathogenesis of Parkinson’s Disease (PD), a neurodegenerative disease characterized by the progressive loss of cognitive functions and motor movements. The mutation in gene glucosidase, beta acid 1 (GBA) is associated with the genetic risk for accelerated PD progression. Prior experiments have shown that GBA deficiency accelerated protein aggregation and affected extracellular vesicles. This has led to our hypothesis that extracellular vesicles are a vehicle for the spread of protein aggregation and mutations in GBA promote this accelerated spread. To investigate this, we developed a Drosophila model of GBA deficiency (GBAdel) and a human neuronal model with induced pluripotent stem cells (iPSCs) from an individual with PD heterozygous for a null GBA mutation. With our fly model, I have conducted genotype recombination to express human Alpha-synuclein (aSyn) in GBA deficient flies in the thorax. If aSyn ended up aggregating in the brain, I compared if this process was accelerated with GBA deficiency versus the control group by measuring high molecular weight oligomers. In addition, prior research has shown that extracellular biogenesis is a result of endolysosomal trafficking which leads to the formation of exosomes in our neuronal culture and spread of protein aggregation in our fly model. We suspect that GBA deficiency affects multiple parts of the endolysosomal pathway. In our human neuronal culture model, we compared endolysosomal trafficking impairments in GBA deficient cells versus controls. To measure this, we stain cells with antibody markers for early endosomes (EEA1,Rab5), late endosomes (Rab11), lysosomes (LAMP1) and conduct confocal imaging for analysis. By understanding the mechanisms of GBA deficiency and progression of protein aggregation, we can determine new therapeutic targets to slow the rate of PD and other neurodegenerative diseases.

Variants in the genetic risk factor GBA have been shown to increase the risk of developing Parkinson’s disease (PD) and accelerate motor and cognitive decline in PD patients. To better characterize this relationship, this project investigates the mechanisms underlying the onset and exacerbation of Parkinson’s disease in patients with the genetic risk factor GBA through the use of Drosophila and human neuronal cell culture models. We use a GBA deficient Drosophila model, which exhibits symptoms of Parkinson’s disease, including neurodegeneration, motor and cognitive dysfunction, and accelerated protein aggregation. Additionally, we use induced pluripotent stem cells (iPSC) from a PD patient heterozygous for the GBA mutation. Prior work in the lab found that GBA deficiency accelerates protein aggregation, alters lipid metabolism, autophagy, and cell-to-cell propagation of pathogenic protein aggregation via extracellular vesicles (EVs). We also found that restoring wildtype GBA function in glial cells of GBA deficient flies rescues protein aggregation in the brain, leading us to hypothesize that GBA may have a neuroprotective role in glia. Because EVs are formed through the endolysosomal trafficking system, we are examining makers for endolysosomal vesicles in GBA deficient and control astrocytes. We will also observe how GBA deficient versus control astrocytes uptake and traffic neuronal EVs, and eventually test whether co-culturing wildtype astrocytes with GBA deficient neurons may reduce pathogenic protein aggregation in neurons, compared to co-culturing GBA deficient astrocytes with GBA deficient neurons, or GBA deficient astrocytes with control neurons. To perform these experiments, we will be using an automated cell culture system integrated with automated confocal microscopy to observe the survival of the cells over time before fixing and analyzing pathogenic protein aggregation in the cells by Western blot. We hope that this research helps us to better understand the mechanisms underlying the progression of Parkinson’s and explore new therapeutic targets. 

Chimeric antigen receptor (CAR) T cells are used to treat blood cancers; however, neurotoxicity is a common complication that can be life threatening. The neurotoxicity patients may experience includes language and cognitive disorders, seizures, and cerebral edema and hemorrhage. In our previous research on a mouse model of CAR T cell toxicity, we found that leukocytes plugged 11.9% of brain capillaries in CAR T cell treated mice along with an increase of Intracellular Adhesion Molecule (ICAM-1) on brain capillary endothelial cells. We hypothesized the effects of capillary plugging may contribute to neurotoxicity, hence to better understand the mechanism, I am exploring the effects of different cytokines we observe in patients with neurotoxicity on ICAM-1 expression on human brain microvascular endothelial cells (HBMECs). I treated cultured HBMECs in 10, 100, and 1000 picograms/mL of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, CXCL10, GM-CSF, TNF-α, and IFN-γ for 24 hours. Then, I used flow cytometry to measure the amount of ICAM-1 and VCAM-1 expressed by HBMECs. Vehicle controls are included, and isotype control, single stains, and live dead stains are used for flow cytometry. I am comparing the change in the median brightness of ICAM-1 and VCAM-1 expression on HBMECs in response to each cytokine. Interestingly, we found IL-1β significantly increases ICAM-1 expression even with doses slightly above normal blood levels (2.5pg/mL). There were significant increases with TNF-α and IFN-γ only at doses reflecting highly elevated levels in the blood (1000 pg/mL), whereas there was no response to high doses of IL-2, IL-6, IL-8, and IL-10, CXCL10, and GM-CSF. Further studies blocking cytokines that greatly induced ICAM-1 expression in a mouse model and test if that will reduce neurotoxicity without affecting effectiveness of CAR T treatments would help us understand the underlying mechanisms of what causes neurotoxicity.

During non-REM slow-wave sleep, the thalamus and cortex generate widespread synchronized epochs of action potential firing that repeat at 1 to 7 Hz. This synchronization is thought to be an essential component of healthy sleep. Cortical excitatory synaptic feedback to the thalamus is required to maintain synchronized firing epochs across the thalamus. A major source of cortical to thalamic feedback comes from a subset of layer 5 (L5) pyramidal neurons (PNs) which occur in the largest numbers in the primary motor cortex. There is also evidence that the basal dendrites of those neurons in the motor cortex receive monosynaptic executory inputs from excitatory thalamic neurons but the number and strength of those connections are not known. For our project, we decided to quantify the number of thalamic inputs onto the basal dendrites of those L5 PNs that project back to the thalamus. I used tissue from the motor cortex of thy1 mice which express a yellow florescent protein in the L5 PNs that send axon branches to the thalamus. The tissue was treated with an antibody to the VGLUT2 protein which is selectively expressed in excitatory synaptic terminals from thalamic neurons. The VGLUT2-containing terminals were visualized using florescent immunocytochemical techniques combined with confocal microscopy to count the number of putative thalamic synaptic terminals that were closely opposed to spines (postsynaptic protrusions) on the L5 PN basal dendrites. Preliminary results suggest that between 5-20% of the dendritic spines are closely opposed to VGLUT2-containing presynaptic terminals. The study of this thalamocortical loop will allow for a better understanding of the processes that are necessary for proper sleep and the implications of disrupted neuron synchronization.