Research

The primary products of the genome are large number of expressed proteins dubbed the proteome, which codifies molecular organization in cells and is a major domain of ongoing scientific inquiry.  Our research centers on identifying and characterizing proteome interaction networks for the dopamine and nicotinic acetylcholine receptors in order to better understand the function and regulation of these receptor systems in various types of cells. Recent projects include:

 

Delineating the role of the nicotinic acetylcholine receptor in synaptic growth and nerve regeneration

Cholinergic neurotransmission plays an important role in brain and spinal cord development shaping synaptic plasticity and guiding regeneration. Various nicotinic receptors (nAChRs) are expressed early in the human brain and spinal cord participating in signals that shape neural connectivity and synaptic wiring. We are interested in the mechanism by which the α7 nACh regulates synaptogensis through its early expression in embryonic neurons of the cortex and hippocampus. This receptor appears genetically linked to various developmental brain disorders including schizophrenia.  We have shown a role for α7-mediated calcium and G protein signaling in the regulation of cytoskeletal motility leading to neurite retraction at the growth cone. Outstanding questions remain on the role of the α7 receptor in shaping synaptogensis in the developing brain.  

α7 and G protein‐regulated inducer of neurite outgrowth 1 (Gprin1) associate in the growth cone. (a) Hippocampal neurons from P0 pups were cultured for 3 DIV. Neurons were probed with fBgtx (green) and anti‐Gprin1 Abs (red) (top images). A heat map measure of the co‐signal (bottom image) shows the distribution and co‐localization of the two proteins in the growing axon and GC. Co‐localization was highest in the soma, growth cone (GC), and branch points (BP) (arrows). (b) Localization of the fBgtx and Gprin1 signals in the GC. CZ: central zone. (c) Protein detection of α7 and Gprin1 within GC fraction (GCF) obtained from P0 pups as described in Materials and methods. Growth‐associated protein 43 (GAP‐43) is used as a marker for the GCF. Cell fraction (CF). (d) An anti‐α7 and anti‐Gprin1 Ab was used to immunoprecipitate (IP) α7 and Gprin1 proteins from the GCF. Western blot detection confirms interaction of the two proteins. Protein identity was also determined by in‐gel digest mass spectrometry (Table S2). IgG was used as an Ab control. From Nordman and Kabbani, 2013

 

Spatio-temporal calcium signal for cytoskeletal growth

Calcium serves as dynamic regulator of biological signaling events within cells owing to its ability to differentially activate various secondary messenger and effector pathways. Spatial and temporal cytosolic calcium level fluctuations are known to encode information for signaling and cytoskeletal motility.  However, it is unclear how calcium transients encode information for processes such as cytoskeletal remodeling during growth and migration. Using real-time calcium and cytoskeletal imaging we are examining interactions between these systems in the growth. In particular, we aim to explore how α7 nACh regulates cytoskeletal growth through calcium entry and store release in caclium microdomains where the receptor is expressed.  

Synaptic α7 nAChRs Couple to Intracellular Stores. Agonist stimulation of presynaptic α7 nAChRs (with ACh or nicotine) promotes membrane depolarization (ΔV) through ionotropic activity (α7i; green). The α7i state is open briefly however, enabling sufficient membrane depolarization via Na+ influx to activate local VGCCs and Ca2+ influx leading to RyR-mediated CICR from the ER in the presynaptic terminal. The transition (τ) of the α7 nAChR into a high affinity ligand-bound desensitized state (α7m; red) supports a metabotropic response marked by G protein activation of PLC, IP3 production, and IICR. The totality of the α7 nAChR calcium transient in the presynaptic terminal is the sum of CICR and IICR, which can modulate neurotransmitter release and synaptic plasticity. From Kabbani and Nichols, 2018.

 

Identification and characterization of receptor interactomes 

A long standing interest has been the delineation of receptor interactomes that underlie receptor signaling and function. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor–protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties in vivo remains a challenge. We are developing tools aimed at examining interactions of the nAChR from neural, neuroendocrine, as well as immune cells. An understanding of nAChR interactomes spearheads molecular biology by enabling the generation of new hypotheses and can lend insight into improving drug development .

Proteomic detection of α7 receptor and G-protein complexes in the brain. A, IP using anti-Gαs/Gαq/Gαi/Gαo/Gβ or anti-α7 antibodies to isolate α7/G protein complexes from 250 μg of mouse hippocampus (1), prefrontal cortex (2), and striatum (3) membrane fractions. Blots were immunoblotted with mAb 306 or anti-Gαq/β antibodies. The blots are representative of 3 separate experiments. B and C, α-Bgtx-binding proteins (BgtxC) were isolated from 1500 μg of a membrane protein fraction of the adult mouse brain. BgtxC was eluted with 1 or 0.5 M carbamylcholine (Carb). B, Western blot detection of α7 within the BgtxC using mAb306. Experiments in α7−/− tissue were used as a negative control. C, BgtxC was divided into 3 molecular weight fractions for proteomic analysis: F1, 190-90 kDa; F2, 89-45 kDa; F3, 44-15 kDa. D, G proteins identified within F2 and F3 fractions. Protein score (Score); molecular weight (MW); and GenBankTM accession number (GI). Total: 10% of the total membrane fraction loaded as a positive control. From King et al., 2015