Microglia, the resident immune cells of the brain, become activated and mediate neuroinflammatory responses in response to traumatic brain injury (TBI). This neuroinflammation can be detrimental to the health of the brain; thus, inhibition of this natural response can benefit TBI patients. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a technological advancement that allow manipulation of specific cellular signaling pathways. The hM4D DREADD gene was inserted in a viral construct downstream of the CD68 promoter, which is markedly upregulated in activated microglia following TBI. This construct is activated with clozapine n-oxide (CNO) to downregulate secondary messengers in activated microglia and thus attenuates the inflammatory response by reducing microglial activation and proliferation after injury. After viral transfection to deliver the construct to cells, rats underwent a controlled cortical impact (CCI) - an experimental model of TBI - and were treated with a CNO injection at varied intervals after injury. After the rat brains were dissected and sectioned, we used immunohistochemistry techniques to label microglia with an anti-Iba1 antibody, proliferating cells with an anti-BrdU antibody, and cell nuclei with DAPI. This allowed for the visualization of microglia using fluorescence microscopy and microglia were quantified using stereological principles. The aim of this research project is to use pharmacological DREADD receptor-mediated inhibition of microglia at different intervals post-injury to quantify the proliferation of microglia following CCI. We hypothesize that increased duration between CCI and CNO injection leads to more pronounced microglial activation represented by increased numbers and morphological changes. We also hypothesize that microglial activation can be observed as a gradient with the greatest proliferation closest to the CCI epicenter.

Charcot-Marie-Tooth (CMT) is a group of inherited nerve disorders resulting in muscle weakness in which the motor and/or sensory peripheral nerves are affected; hereditary neuralgic amyotrophy (HNA) is a rare genetic disorder characterized by severe pain and muscle wasting in the arms. Using whole exome sequencing (WES), we aimed to find the causative mutation in a five-generation family with CMT and single-generation family with HNA. WES is an efficient method to identify possible disease-causing mutations by searching for variations in the protein-coding region of any gene. After excluding variants from a known list of genes to be causative of CMT, we used stepwise criteria to filter and prioritize the candidate variants in the exome data from two patients in this family: heterozygosity, alternative splicing, gnomAD frequency, CADD score, function of the gene, and its expression. In CMT, out of the 26 candidate variants, 10 were INDEL mutations and 16 were single nucleotide polymorphisms (SNPs). Each variant was Sanger sequenced to verify the exome variant; the verified variants were further evaluated for co-segregation with the disease by Sanger sequencing affected and unaffected individuals of the family. The results from these analyses did not identify any candidate variant as a possible candidate, concluding the CMT WES study without yielding a causative gene. Our next steps for this project will be to use whole genomic sequencing (WGS) to reveal DNA variation outside the exome or copy number variant or repeat expansion that may alter protein production to identify potential causative mutations. Currently, we are using the same methodology to screen for candidate variants in three exomes from the family with HNA. Out of 22 candidate variants, we have screened 2 INDEL mutations and 2 SNPs. We are actively screening the remaining variants in search of a causative mutation for HNA in this family.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic event confers neuronal and axonal protection against subsequent ischemic exposure. The cell types responsible for IPC in the brain are unknown. In a novel model of white matter (WM) IPC and ischemic injury, we identified specific innate immune signaling pathways in microglia as required for IPC-mediated axonal protection, leading us to suspect that microglia are required for IPC. The model of WM IPC involves exposing the mouse optic nerve (MON) to a brief ischemic event 72 hours before the MONs are isolated and exposed to oxygen-glucose deprivation. Animals were treated with PLX5622 - a colony stimulating factor-1 receptor (CSF1R) pharmacologic antagonist – to deplete microglia in the central nervous system, including the MON, to test our hypothesis. By recording axonal function, we determined that microglial depletion eliminated IPC-mediated axonal protection in the WM. This study examines the impacts of IPC and ischemia on the nodes of Ranvier, which we hypothesize are protected by preconditioned microglia. The length of the nodes of Ranvier affects conductance and action potential propagation through an axon, with recent publications suggesting elongation of the notes in disease or injury states. We hypothesize the nodes of Ranvier will be shorter in preconditioned MONs than non-preconditioned MONs, which would support IPC-mediated protection. Additionally, we expect the nodes of Ranvier in PLX5622-treated MONS will be of similar length as non-preconditioned control MONs, indicating loss of IPC-mediated protection in animals without microglia. We will use immunofluorescence and confocal microscopy to measure the nodes of Ranvier and correlate these anatomical findings to prior electrophysiology experiments. The results of this research would contextualize a novel understanding of how microglia and ischemia affect the WM in specific regions of the axon, which is vital for advancing the development of neurotherapeutics for stroke.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide and has been established as a risk factor for neurodegenerative diseases such as Alzheimer’s disease (AD). The progression of AD is characterized by intracellular aggregates of phosphorylated tau protein, which is mainly found in neurons and plays an important role in the stabilization of microtubules. One of the mechanisms that may contribute to tau aggregation is decreased tau clearance by the glymphatic system, a pathway that clears solutes from the brain. This fluid movement is facilitated by the astrocytic water channel aquaporin-4 (AQP4) which is primarily localized to the astrocytic endfeet that line perivascular channels surrounding the brain vasculature. Prior studies demonstrate that solute clearance along these pathways is slowed following TBI, and that there is a loss of perivascular localization of AQP4. Based on these findings we hypothesized the loss of perivascular localization of AQP4 may impair interstitial tau clearance and promote neurodegeneration. We first tested this hypothesis by examining whether loss of perivascular AQP4 following TBI promotes tau pathology in a transgenic PS19 mouse that spontaneously develops tau pathology. We then evaluated whether deletion of perivascular AQP4 in an alpha-syntrophin knock-out mouse promotes tau pathology both in the presence and absence of TBI, and when crossed with a PS19 tauopathy mouse. Alpha-syntrophin is a protein that anchors AQP4 and is important in perivascular localization; therefore, deletion of alpha-syntrophin results in loss of localization of AQP4 and impairment of clearance. We assessed levels of pathological tau using histology on the transgenic mice and crosses both with and without TBI. If validated, our findings may suggest that loss of perivascular AQP4 may increase the brain’s vulnerability to tau aggregation and neurodegeneration following TBI and provide the basis for potential treatment to prevent the development of post-traumatic neurodegeneration.

Amyloid β (Aβ) plaques are a hallmark of Alzheimer’s disease (AD), the most common form of dementia that afflicts over 5 million Americans. Soluble proteins, including Aβ, are cleared from the brain by the glymphatic system, a brain wide network of perivascular spaces that facilitates the intermixing of cerebrospinal fluid and interstitial fluid. Prior studies report that aquaporin-4 (AQP4), a water channel polarized to perivascular astrocyte endfeet, supports glymphatic clearance of soluble proteins from the brain. In the aging brain, glymphatic clearance becomes impaired and AQP4 becomes depolarized from astrocytic endfeet. Such loss of perivascular AQP4 localization is correlated with AD status and Aβ plaque burden in the human brain. In the present study, we test whether such AQP4 depolarization promotes Aβ plaque formation. AQP4 is anchored to astrocytic endfeet via the dystrophin protein complex that includes the adaptor protein α-syntrophin (α-Syn). We crossed the α-Syn knockout mouse, which lacks perivascular AQP4 localization, with the 5XFAD mouse line which spontaneously develops Aβ plaques. Our preliminary analysis suggests that loss of perivascular AQP4 localization with α-Syn knockout increases Aβ burden relative to controls. These findings demonstrate that loss of perivascular AQP4 localization, such as occurs in the human brain in the setting of AD, contributes to the development of Aβ pathology. In the future, it may be possible that targeting the localization of AQP4 may be the basis for new therapeutics that can slow or even reverse AD pathology.

Antiseizure medications (ASM) may contribute to adverse fetal outcomes in pregnant women with epilepsy (WWE). Folate processing (methylenetetrahydrofolate reductase, MTHFR) gene abnormalities are common in women with epilepsy and depression. The enzyme product of this gene is a component of the metabolic pathway that makes folate bioavailable through methylation, critical for maintaining adequate serum folate levels. Folate is known to be crucial for fetal development, namely in the prevention of spina bifida and other neural tube defects (NTDs). The teratogenesis of some ASM in combination with genetic polymorphisms put WWE at higher risk for infertility, miscarriage, and/or major fetal malformations. L-methylfolate supplements may bypass deficiencies in the MTHFR-mediated folate metabolism pathway, yet their use in WWE during gestation or on fetal development is not well studied. We hypothesized that supplementation with L-methylfolate and methylcobalamin (methylated B12) may support better perinatal and fetal outcomes in pregnant WWE. We examined pregnancy histories of three WWE who supplemented with either folate or L-methylfolate and methylcobalamin (methylated B12) during pregnancies. Their pregnancy outcomes (both in conception and gestation) as well as mood stability improved with supplementation. L-methylfolate and methylcobalamin supplementation merits further study in WWE who have MTHFR mutations, fertility, recurrent miscarriage and/or depression histories.

Tau is a microtubule associated protein (MAP) that is regularly expressed under normal conditions to stabilize microtubules. However, in several neurodegenerative diseases including Parkinsons, Alzheimers, and epilepsy, tau undergoes excessive post-translational phosphorylation which causes tau to lose affinity for microtubules and become “soluble”. The accumulation of the unbound protein hypothetically promotes aggregate formation, which has become a pathological feature of these neurodegenerative diseases. In animal models of epilepsy, genetic deletion of tau reduces seizure occurrence, and in human epilepsy there is some evidence that phosphorylated tau (p-tau) accumulates in brain tissue. Both of these suggest that downregulation of tau expression and function may protect against seizures. We hypothesized that in an animal model of epilepsy we would similarly find loss of tau expression and increases in p-tau expression. So far, using western blot analysis we have found a decrease in the expression of total tau in the hippocampus (thought to be the focal point of seizures) in a rat model of temporal lobe epilepsy (75.45 ± 3.11 % of age-matched controls; n = 8; p = 0.0014). Also, we are investigating novel and homologous phosphorylation sites for tau when comparing rat brain samples and epileptic human brain samples by mass spectrometry. By uncovering potential tau-mediated pathology of epilepsy via identification of novel tau phosphosites in the disease state in both rats and humans, we may develop biomarkers that may be used to determine hyperexcitable regions in the brains of epilepsy patients.