Leigh Syndrome (LS) is the most common pediatric mitochondrial disease, and it is associated with loss-of function mutations in genes that encode for proteins in Complex I of the electron transport chain. Mutations in a gene called NADH dehydrogenase (ubiquinone) iron sulfur protein 4 (NDUFS4) is linked with LS which results in many neurological symptoms and neurodegenerative biomarkers in afflicted patients. Prior studies have discovered that many neurodegenerative diseases are characterized by disturbances in circadian function, which can impact disease symptoms and worsen quality of life. However, it remains unknown if disruptions of circadian function are a characteristic phenotype of all mitochondrial diseases and Leigh Syndrome in particular. In this study, we investigated the integrity of the circadian rhythm in conditional knockout (KO)-models of LS. KO-models carried the mutation in either excitatory (glutamatergic) or inhibitory (GABAergic) neurons. We generated mice with Ndufs4 KO restricted to glutamatergic or GABAergic neurons using LoxP Cre technology. To examine circadian rhythm patterns, we placed each mouse in an individual cage with a running wheel and infrared (IR) sensor. Mice were maintained on a 12:12 hour light-dark schedule where the light period began at 7:00 AM. Mouse wheel activity and home cage locomotor activity were recorded and subsequently analyzed offline using ClockLab Analysis. Our initial results showed that Ndufs4 KO in excitatory neurons leads to severe disruption of circadian rhythms in which locomotive activity was not synchronized with a light-dark cycle, whereas Ndufs4 KO in inhibitory neurons had no detectable effect on circadian rhythm. These results reveal that disruptions in circadian function are present in Ndufs4-related LS, particularly due to excitatory neurons. A better understanding of circadian rhythm disruptions in LS can lead to further research on a molecular level to discover underlying characteristics of LS and become an identifier for the progression of LS.

Anxiety, a heightened state of arousal without the presence of an immediate threat, can be incapacitating once it reaches a disease state. Located in the extended amygdala, the Bed Nucleus of the Stria Terminalis (BNST) has been implicated in sustained fear states and other anxiety-related conditions. BNST neurons have been shown to be diverse, co-releasing neuropeptides and other neurotransmitters, but the quantification and localization of these neuromodulators is not clear. In order to identify the co-expression of neuropeptides I employed in-situ hybridization in the mouse BNST, where RNA sequences specific to relevant peptides are recognized with a fluorescent probe. Then, I created pipelines that identify and group amplified peptide signals to cells, quantifying intensity and presence, and collect them by co-expression and subregions within the BNST. I hypothesize that co-expression of these peptides is regionally biased. Finally, identifying differences in cell-type distribution has implications for anxiety-behavior and may provide insight towards treatment of anxiety conditions.

Tauopathies are a set of neurodegenerative diseases identifiable by the aggregation of insoluble intracellular deposits of the microtubule associated protein tau. Among others, this class of diseases includes frontotemporal dementia (FTD), Pick’s disease, and supranuclear palsy. Though the precise pathology of tauopathies is poorly understood, most forms are associated with hyperphosphorylated tau and impaired clearance of tau through the ubiquitin-proteasome and autophagosome pathways. We attempt to explore the degradative properties of one specific tau variant, a serine to threonine missense mutation (S356T), which is strongly associated with an early onset behavioral variant frontotemporal dementia (bvFTD). Posttranslational modification at the S356 residue in tau controls its proteasomal degradation. Therefore, we hypothesize that the S356T variant exhibits impaired tau clearance compared to wildtype tau. To test this hypothesis, we use the pharmacological inhibitor Cycloheximide that prevents protein synthesis in both human embryonic epithelial cells (HEK293) and neurons transfected with wildtype and S356T tau protein. Cycloheximide has been shown to inhibit translation by binding to the E-site of the 60S ribosomal subunit and preventing protein elongation. This drug allows us to measure protein degradation without being confounded with new protein synthesis. We expect the amount of tau, relative to total protein, to be elevated in the S356T transfected cells after application of cycloheximide. If our hypothesis is correct, we plan to explore possible mechanisms behind the aberrant degradative properties of the S356T tau variant. By exploring the mechanisms through which tau mutants contribute to neuropathology, we hope to provide molecular insight into tau proteostasis that if successful could offer a promising therapeutic target.

Leptin and ghrelin are two hormones essential to maintaining and regulating energy levels and food intake. These hormones have opposite effects on feeding behavior where leptin suppresses feeding and ghrelin potentiates it. While the roles of these two hormones have been widely researched, their relative effects on distinct neural populations are still largely undetermined. Previous electrophysiological and in vivo imaging experiments have shown that the activity of individual populations of glutamatergic hypothalamic projection neurons are differentially affected by feeding hormones. This data leads to the question of whether or not the projection populations have a bias for leptin and ghrelin receptors that could account for the difference in sensitivity. We hypothesize that Lepr and Ghsr will be expressed at different levels within the different projection populations. To study this, we injected two retrogradely trafficked viruses into the target locations, lateral habenula (Lhb) and ventral tegmental area (VTA), in Mus musculus and performed fluorescent in situ hybridization experiments in the lateral hypothalamus (LHA), dorsomedial hypothalamic nucleus (DMH), ventromedial hypothalamus (VMH), paraventricular nucleus (PVH), and arcuate nucleus (ARC) for viral based fluorophores as well as leptin and ghrelin receptors. The viral expression was imaged using fluorescence microscopy and quantified for within individual hypothalamic neurons. Analysis is currently underway to reveal any differences in fluorescence between the two projection populations and therefore, determine any disparities in hormone receptor expression. The objective of this research is to understand whether differences in expression of Lepr and Ghsr exist within LHb- and VTA-projecting glutamatergic hypothalamic neurons. This study could be indicative of how hormones regulate feeding behavior and their particular effects on the hypothalamus projection neurons. Further research concerning the downstream impacts of these opposing hormonal pathways could shed light on the neural networks that govern food and energy balance.

Loss of neurons in the retina underlie many blinding diseases, such as macular degeneration, glaucoma, and diabetic retinopathy. These retinal neurons are not replaceable. The Reh Lab has developed a strategy to achieve functional regeneration of neurons in the adult mammalian retina, but not enough regeneration to restore vision loss. One of the potential limitations to restoring vision is the inflammation that occurs during retinal injury or disease. All mammalian retinas contain microglia, which are the primary immune cells of the nervous system that respond to pathogens, injury, and disease. My goal is to determine how microglia respond to damage-induced regeneration in the retina. After damaging mice retinas with an excitotoxin (NMDA), we induced neuronal regeneration with a proneural transcription factor called Ascl1. Using immunohistochemistry and confocal fluorescence microscopy, I stained various markers that represent different inflammation states of microglia. I quantified microglia and analyzed their response to neural regeneration. Data from confocal fluorescence microscopy revealed that microglia surround newly regenerated neurons and display a variety of subtypes during regeneration. Follow up single-cell sequencing experiments confirmed that these immune cells show a molecular heterogeneity of states. This study provides a better understanding about how microglia function during retinal regeneration. Results from this study can help reveal targets for manipulation to improve regeneration of the retina. Further work can be done to analyze how these microglia behaviors impact regeneration of lost retinal neurons.