Dravet Syndrome (DS) is a severe form of childhood onset epilepsy occurring in about 1 out of 16,000 births. The disease is characterized by treatment-resistant seizures, ataxia (or loss of muscle coordination), developmental delay, cognitive impairment, and increased rate of premature mortality mostly due to sudden unexpected death in epilepsy (SUDEP). DS is predominantly caused by a heterozygous loss-of-function mutation in the SCN1A gene, which codes for the pore-forming alpha subunit of the NaV1.1 voltage-gated sodium channel. Our lab has previously shown that selectively introducing these mutations into neurons expressing the neurotransmitter GABA, specifically GABAergic interneurons of the forebrain, is sufficient to cause DS phenotypes in mice. In this study, we investigated whether an SCN1A gene replacement therapy precisely targeted to this interneuronal population can rescue epilepsy and SUDEP. Our lab, in collaboration with the Allen Institute, has developed a novel dual SCN1A-intein-AAV with forebrain GABAergic interneuron targeting capability using a Dlx56-based enhancer. Mice treated with this vector at postnatal day (P) 0 via intracerebroventricular injection were monitored for spontaneous mortality up to P70 and tested for susceptibility to thermally induced seizures. All untreated mice (n=31/31) died by postnatal week 6. In addition, 86.5% (n=13/15) of them exhibited thermally induced myoclonic seizures (MCS) and 100% (n=15/15) of them showed generalized tonic clonic seizures (GTCS). In striking contrast, none of the treated mice died (n=9/9, p=3.3e-14, Fisher’s exact test) nor exhibited MCS (n=0/9, p=7.1e-4, Fisher’s exact test) or GTCS (n=0/9, p=1.1e-5, Fisher’s exact test). These findings suggest that precision therapy targeting the very site of disease etiology can completely protect against epilepsy and related mortality in DS.

Rare Intracranial Tumors (RIT) are a heterogeneous group with unmet medical needs. Although infrequent in individuals, RIT affect millions of people who lack effective disease monitoring and treatment. Frequent chromosome gains and losses are common in cancer, leading to the upregulation of oncogenes and downregulation of tumor-suppressor genes, respectively. Somatic Copy Number Variations (CNVs) affect a greater fraction of the genome than single nucleotide polymorphisms (SNPs) and have been correlated to drug resistance and tumor progression, highlighting a potential prognostic value. Cell-free circulating tumor DNA (ctDNA), which are DNA fragments released by necrotic or apoptotic tumor cells can act as a noninvasive cancer biomarker, offering a potential alternative to invasive tissue biopsies. In the present work, we aim to establish the somatic CNV signature of tumor and matched ctDNA to identify non-invasive tumor-related CNVs that may serve as biomarkers for use in liquid biopsy. We performed Whole Exome Sequencing (WES) in gDNA isolated from tumor tissue and matching ctDNA of 15 patients with RITs (pituitary tumor (n=9), craniopharyngioma (n=2), and meningioma (n=4). Raw reads were assessed for quality (Trimmomatic, FastQC), following alignment against human reference genome GRCh38 (BWA). Aligned reads were sorted and subject to duplicate removal using Picard. CNV profiles will be generated using CNVkit tool. Data analysis and visualization will be performed using R and python. The most promising aspects of liquid biopsy in cancer applications are cancer screening and early diagnosis because they can lead to better survival results and less disease burden. At the end of this work, we hope to identify the CNV signatures shared between tumor tissue and ctDNA, provide novel insights into the pathophysiology of these RITs and ultimately, suggest promising biomarker candidates for liquid biopsy.

Leigh Syndrome (LS) is a neurodegenerative disease due to the dysfunction of mitochondria. It usually begins in infancy and its incidence is around 1 in 40,000 individuals. Children with LS experience a progressive decline in their cognitive and motor functions often accompanied by severe treatment-resistant epileptic seizures. Mutations in Ndufs4, the gene that encodes a subunit of mitochondrial complex have been linked to LS. Mice carrying Ndufs4 recapitulate several key characteristic clinical manifestations of LS. Using these mouse models, our lab has demonstrated that GABAergic interneurons play an important role in the pathophysiology of LS. Mice with Ndufs4 knockouts (KO) restricted to GABAergic neurons located across all brain regions exhibit seizures. However, seizures in epilepsy patients and animal models typically originate from forebrain structures. Therefore, in this project, we examined whether the inactivation of Ndufs4 in GABAergic neurons of the forebrain alone is sufficient to cause seizures in mice. Homozygotes floxed Ndfus4 mice were crossed with Dlx56Cre+ or Gly2TCre+ mice to KO the gene specifically in interneurons of the forebrain or brainstem. We hypothesized that only mice with KO in the former region will exhibit seizures. Conditional KO mice from these two lines were tested for thermal seizure susceptibility. Surprisingly, both Dlx56creKO and Gly2TCre KO mice exhibited thermally induced myoclonic and generalized tonic clinic seizures. These findings indicate that GABAergic interneurons regions outside of the forebrain are critically involved in the pathogenesis of epilepsy in LS.

Leigh syndrome (LS) is the most common form of mitochondrial disease in children. It affects 1 in every 40,000 births and its clinical manifestations include ataxia, seizures, failure to thrive and premature death. Genetic mutations in more than 75 different genes 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. Surprisingly, new KO mice with Ndufs4 inactivation restricted to GABAergic neurons of the brainstem and cerebellum interneurons, mediated by GlycineT2Cre, also have epilepsy. In this study, we sought to uncover the brain regions that house neurons involved in seizure activity in these mice. Brain regions experiencing neuronal hyperactivity during seizures in this new model of LS were examined. A thermal seizure was induced in the Ndufs4 GlycineT2Cre KO mice. For control condition, mice were exposed to a sham experiment. Forty-five minutes after the seizures or sham procedure, the mice were anaesthetized, and their brains were fixed and harvested. Brain slices were prepared and stained with a c-Fos antibody and finally imaged on the confocal microscope. Interestingly, high c-Fos immunoactivity was observed in the cerebellum alone and not in forebrain 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 plan to increase the sample size and confirm the results with statistical methods.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects various cognitive functions. Progressive neurodegeneration and disruption of normal brain function can arise due to accumulation of neurofibrillary tangles (tau) in neurons within the entorhinal cortex and hippocampus, driven by a burden, or buildup of amyloid beta (ABeta) plaques. Motivating the present work, those who have experienced traumatic brain injury (TBI) have an elevated risk of developing AD. Our previous study found that low-intensity focused ultrasound (LIFU) reduced the ABeta burden, in a mouse model of AD. Additionally, studies have shown that enhanced activation of cholinergic pathways (therapeutically, by the drug Idazoxan) may be required for improved ABeta clearance and, subsequently, reduce tau accumulation. We therefore sought to test that LIFU (alone or with Idazoxan) can improve brain function after TBI, using a 3XTG AD mouse model expressing both ABeta and tau. After induction of a controlled cortical impact (CCI), we applied treatment to four cohorts of 3XTG AD mice while testing their visual-spatial working memory via a weekly T-maze alternation test. Treatment conditions consisted of: LIFU alone; Idazoxan alone; LIFU + Idazoxan; and CCI only (sham) for four weeks post-injury, whereafter the animals’ brain tissue was collected for protein quantification. I specifically carried out a number of the aseptic (minimal contamination), survival surgeries to create a mouse model of TBI, administered LIFU and Idazoxan treatment, and performed computational analysis of protein burden and behavioral testing results. We observed that AD mice treated with LIFU performed significantly better in the T-maze alternation test at 4 weeks post-injury, despite no change in their ABeta burden as well as significant increase in the phosphorylated-tau/total tau burden. The finding of this study challenges prevailing theories of Alzheimer’s intervention, which largely focus on reducing ABeta burden as a means of reducing tau burden.

Spinal cord injury (SCI) is a prevalent human trauma that greatly reduces an affected individual’s quality of life. Natural healing post-SCI results in glial and fibrotic scarring, which are common bodily reactions to central nervous system (CNS) injury, but components within these scars unfortunately inhibit axonal regeneration and the sprouting of injured neurons. Interestingly, mammals of the genus Acomys have evolved mechanisms to overcome these deficits and regenerate CNS cells to full functionality post-SCI. Since adult mammalian neurons do have an intrinsic capacity to regenerate, we reasoned that neuron-extrinsic factors are the likely culprit for failed cell regeneration in SCI patients. Thus, the overall aim of our project is to characterize neuron-extrinsic factors such as immune cells and alterations in extracellular matrix (ECM) molecules that give the species Acomys cahirinus their regenerative capacity. We hypothesized that Acomys will exhibit greater axonal regeneration and less fibrotic scarring than the common mouse model, Mus musculus, after SCI. To study this, we produced a clinically relevant contusion SCI in both Mus and Acomys. We then examined major inflammatory cells known to be activated post-SCI. Standard immunohistochemistry targeting IBA1 (a microglia marker) and GFAP (an astrocyte marker) was used to detect: 1) microglia, the first-responders to injury in the CNS and 2) astrocytes, a major component of the glial scar. My analysis showed that more microglia, but less astrocytes, were activated in Acomys than Mus. This suggested that after SCI, Acomys activated less astrocytes but recruited more reparative immune cells compared to Mus. Next, we will examine the distribution of fibroblasts and collagen, important ECM components that compose fibrotic scars, in post-SCI Acomys and Mus tissue. Understanding how neuron-extrinsic factors respond to SCI in Acomys will help us further identify and define cellular targets for the development of novel therapeutics to treat human SCI.

 Alzheimer’s Disease (AD) has well-known brain alterations such as Tau protein build-up, beta-amyloid plaques, and neuronal cell death, yet the role of the brain’s microvasculature on the progression of this neurological condition has not been fully uncovered. My research examines changes in the microvasculature density, length, and function during normal aging using a well-established aging model in Brown Norway rats. This study contributes to the pantheon of previous microvascular research and forwards the field toward understanding AD development from another perspective. My hypothesis is that the density and length of these microvasculature are decreased in areas associated with learning and memory (i.e., the hippocampus and parietal cortex) before the development of AD symptoms and worsen as the disease progresses. To test this hypothesis, first in normal aging, 3 experimental groups of Brown Norway rats are employed: (n=6) young rats at 5-6 months, (n=6) middle-aged rats at 15 months, and (n=6) old rats at 20-24 months. Sagittal slices of the right hemisphere were fluorescently marked for their microvasculature, astrocytes, and cellular nuclei. The ImageJ analytical program was used to compartmentalize the areas of the dentate gyrus, CA1, CA2, and CA3 along with the parietal cortex into 900 x 900-pixel boxes for examination. The preliminary results show that the density of microvasculature within the 3 age groups were consistent while the distribution of the vessel lengths had more variability. The two leading postulates are increased tortuosity with increased age and/or rarefaction, where the microvasculature experience shortening with increased age. Further analysis is needed to examine this distribution among the 3 age groups.

Neurogenic bladder is a common condition associated with traumatic spinal cord injuries (SCIs), which results in inhibited detrusor function, bladder-sphincter dyssynergia, and scarring of the bladder walls and muscle. Care for neurogenic bladder is aimed at reducing abnormally high pressures, which left untreated lead to hypertrophy and tissue fibrosis of the bladder wall, as well as upper urinary tract complications. Current treatments target the neurotransmitter release of acetylcholine, utilizing anticholinergic drugs to counter the overactive bladder. However, these drugs can have negative/deleterious side effects, and have broad symptoms influencing unintended targets around the body. Targeted chemodenervation that uses Botulinum toxin (Botox) to interfere with nerve conduction is often reserved as a second line of defense to treat neurogenic bladder. Unfortunately, this late provision concedes irreversible damage to the detrusor muscle. We hypothesize that administering early chemodenervation can prevent the development of neurogenic bladder, improve bladder compliance post SCI, and increase the overall quality of life of SCI affected patients. Using a rat model, the Khaing lab administers a controlled contusion injury to the T8/T9 vertebrae, simulating a spinal cord injury in humans. The recovery of the rats is tracked with behavioral observations, cystometry data collection, and histological stains. My team and I are working to determine the effective therapeutic time post SCI for Botox injections, and the optimal doses for treatment. Our results thus far support that acute chemodenervation with Botox reduces bladder overactivity, and bladder wall thickness.