Cellular and Molecular Brain Research Laboratory [CaMBR]

Research - Overview

Research projects in our laboratory focus on the cellular and molecular mechanisms that are associated with neurodegenerative diseases, with particular emphasis on Alzheimer’s disease (AD) and traumatic brain injury (TBI). Our laboratory projects span from investigations of basic mechanisms underlying AD pathogenesis and investigations of the mechanisms that confer increased risk for developing AD to translational biomarker discovery projects in patient samples. Specific projects include the investigation of the role of γ-aminobutyric acid GABA (B) receptor function in AD, mechanisms underlying how diabetes confers increased risk for developing AD, and chronic neuroinflammation in AD and TBI pathogenesis. Our research involves several pre-clinical transgenic mouse models, extensive cellular and molecular biology approaches that range from cell culture to flow cytometry and mass spectrometry. We employ these same techniques to human samples in our biomarker discovery work to investigate potential mechanisms of AD pathogenesis. Our laboratory is divided into two interrelated arms consisting of the Cellular and Molecular Brain Research (CaMBR) Laboratory that focuses on our pre-clinical mechanisms research and the Pam Quirk Brain Health and Biomarker Laboratory) in which we pursue the investigation of candidate biomarkers for our own specific projects, as well as serves as the biomarker discovery arm for several collaborations and funded Center grants.

GABA:

GABA is the primary inhibitory neurotransmitter in the brain, and plays a vital role in many aspects of neuronal functionality. We are particularly interested in the role of the metabotropic GABA B receptors. These receptors are responsible for slow and sustained inhibition that are heavily involved in learning and memory. Post-mortem investigations of brains with Alzheimer's disease and schizophrenia indicate altered levels of this particular receptor and associated proteins, suggesting a possible role of the GABAB receptor in the progression of these diseases.

In our 2015 publication: "Postnatal alterations in GABAB receptor tone produce sensorimotor gating deficits and protein level differences in adulthood" published in International Journal of Developmental Neuroscience, we demonstrated an effect of altered GABAB receptor signaling on learning and memory. The administration of baclofen –  GABAB agonist –  produces a robust increase in freezing behavior after the acquisition of cued and contextual fear conditioning. Further, the administration of this ligand prevented the extinction of contextual fear association. These data may indicate a role for GABAB receptors in updating previously learned associations. Additionally, in some of our other GABAB-related studies, baclofen was administered during early life (on postnatal days 7, 9, and 12) and produced robust alterations to GABA signaling during adulthood. 

With our findings involving GABAB in learning and memory, we investigated the role of this receptor in Alzheimer’s disease pathogensis. Our most recent publication demonstrates alterations in GABAergic signaling in a well-established mouse model of Alzheimer’s disease, the APP/PS1 (Salazar 2020). The study evaluated 3 timepoints, 4 months of age, 6 months of age, and 9 months of age to evaluation changes though the progression of the disease. We used the Barnes maze to assess learning and memory. In addition, tissues were evaluated through a number of cellular and molecular techniques, including western blot, qRT-PCR, and immunohistochemistry. Changes were identified in GABAB receptor subunits, as well as GABA-related proteins. With GABAB receptors expressed on both neurons and glia (See figure), as well as the significant changes of GABAB in with the presence of amyloid pathology, these findings represent a novel mechanism altered in Alzheimer’s disease that impacts neuronal and glial function.

Figure 9 from our 2020 manuscript "Alterations of GABA B receptors in the APP/PS1 mouse model of Alzheimer’s disease" published in Neurobiology of Aging depicts localization of GABA B2 receptors on the microglia in vivo (6 months old). Aβ plaques (green) were surrounded by microglia (red). GABA B2 receptors (pink) were present in the entire section that indicated their presence in the neurons and in the microglia. GABA B2 localized at the surface of the microglia. Images taken at 100x magnification.

Diabetes

Diabetes Mellitus is a major risk factor for developing Alzheimer’s disease (AD), as diabetics confer up to 4 times increased risk for developing AD, however, the underlying mechanisms are poorly understood. We are currently investigating the role of diabetes that are associated with the onset of sporadic Alzheimer’s disease in both preclinical and clinical models and samples, respectively. Among these risk factors are disruptions to insulin signaling, hyperglycemia, chronic neuroinflammation, and ageing. We investigate these risk factors independently, as well as collectively, to better understand how they might interact and promote learning and memory deficits and exacerbate AD pathogenesis. We are currently utilizing streptozotocin (STZ), a compound that induces hyperglycemia, by infusing directly into the brain (ICV) and/or peripheral administration, to understand how the different roles of insulin in the brain and in the systemic body can lead to common pathways that may underlie the onset of Alzheimer’s disease. 

In our 2016 manuscript titled “Effect of acute lipopolysaccharide-induced inflammation in intracerebroventricular-streptozotocin injected rats” briefly, we found that STZ-ICV elevated levels of Aβ peptides and phosphorylated tau, both pathological hallmarks of Alzheimer’s disease. Additionally, we activate the immune system, usually using lipopolysaccharide, in order to investigate the role of both acute and chronic inflammation to better understand the contribution of microglia and the immune response in exacerbating existing AD-like pathologies.

In our 2018 publication titled “Intermittent streptozotocin administration induces behavioral and pathological features relevant to Alzheimer's disease and vascular dementia” briefly, we found that chronic hyperglycemia induced via Intermittent peripheral streptozotocin, induced a sustained hyperglycemic state in an otherwise completely healthy animal, increased inflammatory signaling as well as elevated hyperphosylated tau  and learning and memory deficits, and alterations in insulin and insulin signaling mechanisms, as well as evidence of microvascular hemorrhages.

Cell Culture – Mechanism investigations

Our lab is currently investigating the role of G protein-coupled receptors (GPCR) on microglial cell populations, specifically in relation to neuroinflammation and AD. To do this, we isolate primary microglia from a novel transgenic mouse line and compare them to microglia isolated from a wildtype (C57BL/6J) mouse. We employ several assays to examine cell functions such as motility and phagocytosis. In addition, the lab utilizes immortalized murine microglia (BV2). These cells are transfected with an oncogene allowing them to divide every 24 hours making it easy to generate a large number to perform different experiments. One such experiment is activating a GPCR pathway using a drug agonist specific to a receptor and subsequently exposing the cells to an inflammatory agent such as lipopolysaccharide or polyinosinic:polycytidylic acid (poly I:C). From then, we can measure the mRNA expression of inflammatory agents called cytokines as well as the level of receptor expression through the use of Quantitative Real-time PCR (qRT-PCR), allowing us to investigate how GPCR role in microgliosis seen in both preclinical and clinical samples.