Leigh Syndrome (LS) is the most common form of mitochondrial disease in children. It affects 1 in every 40,000 births and is characterized by ataxia, seizures, failure to thrive and premature death. There are more than 75 gene mutations that have been associated with LS. Among them is NDUFS4, the gene that codes for a subunit of the protein complex I of the mitochondria. Mice carrying a whole-body knockout (KO) of this gene greatly model this illness; they recapitulate multiple phenotypes of LS in patients. Prior studies in the lab have shown that the KO of Ndufs4 in GABAergic neurons, not in excitatory neurons, across all brain regions, reproduce the epilepsy phenotype seen in the global KO mice. Moreover, GABAergic neurons in a specific brain region such as the brainstem are sufficient to lead to epilepsy in mice. Mice with Ndufs4 KO in brainstem and cerebellum interneurons, mediated by GlycineCre, have epilepsy. However, it is still unclear as to what brain regions housed neurons involved in seizure activity in these mice. In this study, brain regions experiencing neuronal hyperactivity and hypersynchrony during seizures in this new model of LS were examined. A thermal seizure was induced in the Ndufs4 GlycineCre KO mice. Forty-five minutes after the seizures, the mice were anaesthetized, the brains were fixed, and harvested. Brain slices were prepared and stained with a c-Fos antibody and finally imaged on the confocal microscope. Surprisingly, high c-Fos immunoactivity was observed in the cerebellum alone and not in other brain regions generally known to be involved in seizure generation. These findings indicate the participation of the cerebellum in seizure generation in Leigh Syndrome epilepsy. In future studies, we will repeat this experiment to increase the sample size and confirm these findings.

The Ndufs4 gene codes for a subunit of complex 1 in the mitochondrial respiratory chain. Mutations causing a loss-of-function of the Ndufs4 gene are linked to Leigh syndrome(LS), a progressive neurodegenerative disorder. LS is characterized by progressive loss of mental, and movement abilities, respiratory distress, and epilepsy, leading to premature death within 2 to 3 years. It is the most common clinical pediatric presentation of mitochondrial disease. Mice with global Ndufs4 knockout(KO) are a good model of LS, exhibiting seizures and other phenotypes of LS. Previous work has shown that epileptic seizures arise from the impact of Ndufs4(KO) in GABAergic interneurons. The objective of my project is to further our understanding of the mechanism of LS-related epilepsy. I assessed the impact of the Ndufs4(KO) on interneuron morphology as a possible contributing factor to the development of seizures. The LS mouse model was generated using the Cre/LoxP mechanism. Two mice with floxed Ndufs4, one is Dlx12cre positive and the other is homozygous Ai14, were crossed creating mutant mice. Control mice were generated similarly, but parents are Wild-Type (WT) for Ndufs4. Mice were perfused with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). I made 50-um coronal brain slices using the Leica VT1000S Vibratome. Then, I mounted the slices and used the Olympus slide scanner to collect fluorescent images. We found scattered labeling of GABAergic neurons in both the control and mutant forebrains. Our results show that the Dlx12cre+ mouse can label a dispersed population of GABAergic neurons across multiple brain regions. Neural dendrites are visible in our images of control and mutant mice. Optimizations are needed to improve the image resolution for optimal Scholl analysis. This study elucidates the impact of Ndufs4 on interneuron morphology and the contribution of this neuronal characteristic in the mechanism of epilepsy in LS.

Aging is associated with shifts in the composition of brain extracellular matrix chondroitin sulfate glycosaminoglycans (CS-GAGs). CS-GAGs are comprised of repeating glucosamine and N-acetylgalactosamine units that are either non-sulfated (0S-CS), mono-sulfated (4S-CS, 6S-CS), or di-sulfated (2S6S-CS, 4S6S-CS, 2S4S-CS/Dermatan) and participate in the regulation of brain plasticity. The mono-sulfated 6S-CS isomer is predicted to play a key role in the induction of circuit plasticity during neurodevelopment. Therefore, we asked whether this isomer also shows consistent age-related changes between wild-type mice and humans in the regions of the hippocampus and cortex. Our preliminary data generated from cohorts of mice ranging in age from 7 days to 2 years (50%M/50% F) reveal that 6S-CS abundance is highest at 7 days of age and declines with increasing age (9-22 mice/group). We analyzed the relative abundance of the 6S-CS isomer in n=57 hippocampal and cortical human tissue samples (age: newborn - 95 years, sex: 50%M/50% F). Initially, the human samples exhibited the highest abundance of 6S-CS isomer following birth (<1 month age) that then declined at >1M to 29 years of age, phenocopying the results from mice. However, in contrast to mice in which 6S-CS abundance decreased progressively with aging, we found that in humans, 6S-CS abundance began to increase starting at 30 to 99 years of age (R² = 0.84, p-0.0001). The biphasic model of changes in 6S-CS abundance in humans throughout normal aging was previously unknown. Collectively, these findings demonstrate that age-associated changes in brain extracellular matrix 6S-CS isomer abundance in human tissue do not reflect the age-related decline of 6S-CS isomers that occur in mice. Therefore, additional research is needed to establish the utility and robustness of using rodent models to study aging and other age-related extracellular matrix diseases in humans.

Immunofluorescent images are a common way to analyze cell response in the presence of brain disease. Microglia - the brain's immune cells - have a range of functional states dependent on their local environment to keep the brain environment healthy. Microglia are typically stained and viewed with immunofluorescent imaging to study the brain's immune response. Microglial functionality and microglia morphology (shape) are highly correlated [5]. By taking and quantifying images of microglia in healthy and diseased brains, we can gain insights into their functional state and their local environment. In addition, most fundamental research about microglia involves the use of animal models, where many species are used to model brain disease. However, limited research directly compares microglia response in one species to another. Previously, research within the Nance Lab has focused on quantifying rat microglial features such as area, perimeter, or circularity [3]. Here, we developed a method to quantify features of microglia, with a focus on microglial branching – the arm-like protrusions from the cell body expanding upon previous work by adding additional branching features to the quantification pipeline to look at the number and length of branches around each cell, which gives us information on the functional state of the cell. We investigated the species-dependent effect on the microglial shape by analyzing images of cells obtained from the neonatal human-term equivalent rat (postnatal day 10, P10), ferret (P21), and mouse (P12). We see qualitative differences in morphology, such as more extensive branching in the rat compared to the ferret. Our ongoing work aims to quantify feature differences in microglia between the rat and ferret and expand to other species.

Hypoxic Ischemic Encephalopathy (HIE) is an injury to a newborn that can occur during pregnancy or the birthing process. HIE is caused by a restriction of blood flow to the brain which leads to inflammation and neuronal death. A myriad of symptoms and disorders including developmental delays and cerebral palsy can result from HIE. The widespread treatment for HIE among clinicians is therapeutic hypothermia; however, this treatment method can only reduce the severity of the injury, is not a curative procedure, and is only effective in a small percentage of babies with HIE. One possible solution to the lack of effective HIE therapies is a nanoparticle-based therapeutic used to decrease inflammatory responses in the brain following HIE. Prior work in the Nance Lab has shown a polymer nanoparticle can specifically target microglial cells in a pro-inflammatory state in an injured brain. In this study, I have successfully loaded the antioxidant N-Acetyl-Cysteine (NAC) into the polymer nanoparticle platform. Using an ex vivo model of neuroinflammation, I displayed that polymer nanoparticles loaded with NAC can be harnessed to change microglia in a proinflammatory state to a more anti-inflammatory phenotype. This nanoparticle treatment was also combined with a priming dose of azithromycin (AZ) prior to NAC-encapsulated nanoparticle administration to further reduce the rate of microglial inflammation in the injured brain. The reduction of microglial inflammation and increase in anti-inflammatory microglial phenotype presence caused by these two therapeutics provides a platform that can be tested in preclinical models of HIE, with the long-term goal to improve the quality of life for children and families affected by HIE.

Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood and oxygen flow to the brain, is the leading cause of morbidity and mortality in newborns, and currently has no cure. Our lab is investigating curcumin for use as a neuroprotectant agent, as it has anti-inflammatory, antioxidant, and antiapoptotic effects. Our current studies have been focused on further improving the drug encapsulation of curcumin in a polymeric nanoparticle platform, as well as methods to increase long-term shelf-life stability of the nanoparticle therapeutics. Previous research from our lab has successfully loaded curcumin into poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles as a delivery vehicle. PEG-PLGA is an FDA approved, biodegradable polymer platform that allows for improved drug delivery efficiency, controlled and sustained drug release, and improved penetration and diffusion in the brain. We have shown that curcumin loaded PEG-PLGA nanoparticles have resulted in significant neuroprotection when used as a treatment for hypoxic-ischemic neonatal rats (term equivalent to human). In order to progress towards scale-up and clinical translation of the therapeutic, I have tested variations to the formulation method at every step of the formulation process, including changes in PEG-PLGA molecular weight ratios, surfactants, and organic solvents used. I have assessed the impacts of each formulation parameter on colloidal stability and drug loading, with the aim to create a scalable, stable platform that can retain drug delivery and drug activity properties during distribution and shelf-life storage. I have identified that nanoparticle drop size, surfactant type and concentration, and freezing protectant (cryoprotectant) have the biggest impact on drug loading and stability. Improving the stability is the first step in making the therapeutic more accessible, cheaper, and easier to transport for a larger impact.