Stroke accounts for more than half of all patients hospitalized for acute neurological disease in the United States, and is the second most common cause of death worldwide. Microglia, the brain’s specialized tissue macrophages, play a major role in the innate immune response to many neurological diseases including stroke. Ischemic preconditioning (IPC) is a brief period of cerebral ischemia that confers robust but transient tolerance to subsequent ischemic challenge. Elucidating the mechanisms of IPC is a critical challenge in stroke research. A major focus of investigation in the Weinstein lab is the cellular and molecular mechanisms of IPC with a particular emphasis on elucidating the role of microglia in this experimental phenomenon. The Weinstein laboratory has demonstrated using ex vivo flow cytometry that IPC induces a robust increase in the number of microglia that can be identified and sorted from the preconditioned (ipsilateral) cortex. Immunofluorescent microscopy and quantitative stereology on preconditioned mouse brain confirmed that IPC-induces a true in situ increase in the number of microglia. Recent efforts have sought to discriminate whether the origin of this in situ increase lies in cellular recruitment or cellular proliferation. My project has been to use immunofluorescent microscopy to evaluate the extent of double-staining present in preconditioned cortex when using antibodies targeted against cellular markers for microglia (Iba-1) and proliferating cells (Ki-67). Initial findings suggest that the increased counts observed in preconditioned cortex are the result of microglia cell proliferation. We have also begun to employ a protocol using Bromodeoxyuridine (BrdU), a stably incorporated cellular proliferation marker, followed by immunofluorescent microscopy/stereology to quantify the time course of microglial cell proliferation following IPC.

The central nervous system is protected by a specialized population of glial cells called microglia. In addition to other crucial functions, microglia function as resident macrophages of the brain and mount a coordinated response when activated by infection or injury. Although acute inflammation is a necessary component of repair after injury, chronic inflammation can have neurotoxic – rather than neuroprotective – effects. The dysregulation of microglia is associated with many neurodegenerative diseases and is an impetus for investigating the molecular mechanisms that regulate microglial changes in aging and their responses to neural disease and injury. The Garden Lab has previously identified a pathway by which reactive oxygen species or DNA damage modulate microglial anti-inflammatory functions through induction of the p53 transcription factor. Transcriptional activation by p53 leads to increased expression of microRNA’s that suppress the anti-inflammatory transcription factor, c-Maf. The goals for my project are to investigate how c-Maf influences microglial activation states and how changes in the environment influence the expression of c-Maf. In order to assess how c-Maf influences microglia activation, I quantified phagocytic activity in primary murine microglia by flow cytometry in conditions where c-Maf is overexpressed or knocked down. Overexpression or knock-down of c-Maf was accomplished by treatment with adeno-associated viruses (AAVs) containing CMV-promoter driven c-Maf or c-Maf-shRNA, respectively. I also evaluated microglia expression levels of pro-inflammatory and anti-inflammatory activation markers by qRT-PCR following modulation of c-Maf expression or treatment with cytokines. I observed that c-Maf overexpression resulted in increased expression of anti-inflammatory markers and decreased expression of pro-inflammatory markers, and vice versa for c-Maf knock-down. Through this project, we have a better understanding of the influence of c-Maf and inflammatory cytokines on the molecular regulation of microglial functions, and their roles in the pathogenesis of neurodegenerative disease.

Chronic epilepsy is attributed to dysfunctional ion channels, including hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which are regulated by a signal cascade mediated by p38 mitogen activated protein kinase (p38 MAPK). Previous researchers in the Poolos Lab manipulated this cascade and discovered an increase in activation of c-Jun N-terminal kinase (JNK), seen by increased phosphorylated JNK in animals experiencing seizures. This is a novel connection and my project explores the timeline of JNK activity during the development of epilepsy. I hypothesize that JNK activity precedes epilepsy onset and may be causal of chronic epilepsy. To study epilepsy, we administer research-bred rats a pilocarpine injection that induces status epilepticus (SE), a state of continuous seizures. After one hour, the rat receives a phenobarbital injection to halt SE. Seizures begin around one week post-SE and achieve steady-state frequency around six weeks. I analyze brain tissue taken after one hour, one day, and one week post-SE using Western blotting to observe JNK differences from pilocarpine-treated rats to control rats. There are three isoforms of JNK; JNK 1 and 3 run on a 46 kDa band and JNK 2 runs on a 54 kDa band. JNK 3 is thought to have most significance in modulating brain neuronal activity. I have discovered a significant increase of the 54 kDa band at one hour post-SE (130 ± 10%, p<0.05, n=8) and one day post-SE (129 ± 11%, p<0.05, n=11); however, the 46 kDa band was insignificantly changed at both one hour post-SE (104 ± 7%, p>0.5, n=9) and one day post-SE (120 ± 12%, p=0.11, n=11). Gathering data at one week post-SE will show us if JNK activation is potentially causative of epilepsy. By understanding JNK activation, we can explore ways to prevent epilepsy development or lessen its severity after brain injury, through antiepileptic drugs and other therapeutic options.

The striatum encodes voluntary movements and cue dependent behaviors, utilizing dopamine, GABA, and acetylcholine to modulate cortical input based on experience. Parkinson’s disease is characterized by a progressive loss of dopamine that reduces striatal function. This degeneration impacts the function of striatal tonically-active cholinergic interneurons (ChIs) that are the sole source of striatal acetylcholine. A main treatment for Parkinson’s disease is L-Dopa, which partially restores synaptic dopamine. Unfortunately, most Parkinson’s disease patients eventually develop L-Dopa-induced dyskinesias (LIDs). The cause of LIDs is unknown and there is no effective treatment.  Our lab and others have shown that ChIs fire in tonic, burst, and pause patterns. To determine how ChIs might respond to a progressive reduction in dopamine availability that emulates Parkinson’s disease, we generated mice that have a knock-in of the human diphtheria-toxin (DT) receptor (DTR) into the mouse Slc6a3 locus, which encodes the dopamine transporter (DAT). DT injection into DAT-DTR mice causes robust ablation of dopamine neurons, while leaving dopamine neurons in DT-treated wild-type (WT) mice [WT(DT)] intact. Preliminary electrophysiological experiments revealed that dopamine-depletion in these mice causes a reduction in firing that is generated by abnormal intrinsic hyperpolarization-activated cation (HCN) channels. We hypothesized the DA sensitive autonomous activity of the ChIs plays a critical role in the development of LIDs.  We generated and harvested the striata from diphtheria-treated DAT-DTR mice (n=10) and WT(DT) mice (n=10) 14 days following DT, when performance on the rotarod was reduced. Comparison with acute dopamine depletion was determined using reserpine (n=10) and saline-treated mice (n=10), sacrificed 13 hr after injection. Mice were crossed with RiboTag^+/+ x ChatCre^+/- mice which allowed collection of ribosomal-associated mRNA from ChIs following immunoprecipitation. Expression levels of choline-acetyltransferase and HCN 1-4 were determined using an optimized RT-qPCR reaction. Outcome will determine the mechanism underlying LIDs and lead to viral-mediated treatments.