Brain stimulation has emerged as a novel treatment for stroke, a prevalent cause of death and disability worldwide. Studies in rodent models have shown that post-stroke electrical stimulation results in plasticity and neuroprotective benefits. However, techniques that were effective in rodents have rarely translated into clinically viable therapies in humans due to the significant differences in rodent and human neurophysiology and anatomy. Therefore, the goal of our study is to obtain clinically relevant outcomes that describe the mechanisms of stimulation induced plasticity in non-human primates. We combined electrophysiology and immunohistochemistry to investigate the degree of stimulation-induced plasticity and network dynamics after photothrombotic stroke in 4 macaques. We quantified the expression of two biomarkers, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43) in cells within ~10mm from the lesion penumbra. Since PSD-95 is important for the maturation of excitatory synapses and GAP-43 is involved in axonal branching and elongation, evaluating the expression of these two proteins around the lesion core allowed us to compare post-stroke synaptic and axonal plasticity in 2 control and 2 stimulated monkeys. Based on wide-field epifluorescence imaging, we identified the distance from the lesion penumbra at which there was a distinct difference in biomarker immunoreactivity in control and stimulated animals, and performed high-magnification confocal imaging to further investigate the structure of biomarker expression. Furthermore, analysis of the electrocorticography signal showed a largescale downregulation of neural activity following electrical stimulation, while Nissl staining revealed that stimulated monkeys had smaller lesion volumes than controls. These results indicate that stimulation elicits changes at both neurophysiological and cellular level, and may exert a neuroprotective effect on the post-stroke network by reducing metabolic energy consumption.. Therefore, this study investigates the effects of electrical stimulation on neuroplasticity and protection following injury, which may have a profound impact on future therapeutic interventions for stroke.

Tuberculosis (TB) is one of the leading causes of death from infectious disease worldwide. Macrophages are the primary replicative niche and provide critical host defense against Mycobacterium tuberculosis (Mtb), the causative organism. A TOLLIP deficiency is associated with an increased risk of TB in human studies and mouse models. TOLLIP is a ubiquitin-binding protein that interacts with toll-like receptors (TLR) involved in modulating inflammatory signaling. TOLLIP has four critical domains: a ubiquitin-binding CUE domain, a membrane binding C2 domain, an autophagosome interacting motif (AIM), and a Tom1-binding domain that participates in endosomal sorting. However, the domains required for TOLLIP’s immune regulatory function during Mtb infection in macrophages are uncertain. To understand the role of selected domains on TOLLIP’s function, we are developing lentiviral vectors to insert TOLLIP with selected protein domains deleted, along with green fluorescent protein (GFP) to tag transduced cells. We measured viral load and transduction efficiency in HEK cells by analyzing GFP fluorescence through microscopy and flow cytometry. We are currently transducing TOLLIP fragments, whole TOLLIP, and control genes into macrophages lacking the TOLLIP gene. TOLLIP (-/-) bone-marrow derived macrophages (BMDM) were infected with lentivirus after 3 days and stimulated with LPS (10ng/ml) after 5 days. Viral supernatants were collected to evaluate TOLLIP gene expression through fluorescent microscopy. The approaches developed in this project will provide the foundation for understanding the critical structural elements of the TOLLIP protein and their role in preventing TB disease.