Alpha 7/GPRIN1 To Infinity And Beyond

         Our brain's wiring makes us who we are. Without a doubt, this process affects all aspects of our lives. The brain lays the foundation of our neural pathways during development. What structures are responsible for this incredible task? What symptoms would occur if they are damaged? What can we discover from researching the processes involved?

Jacob Nordman, Ph.D

         On November 1st, 2012, George Mason University's Neuroscience Ph.D. Candidate from Dr. Kabbani's lab, Jacob Nordman, presented his research regarding alpha 7 nAChR and Gprin1 to our NEUR410 course. The lecture focussed on alpha 7 and Gprin1 interaction which regulates axon growth and growth cone dynamics on hippocampal neurons. The objective of Kabbani's lab and Nordman's research is to study the proteome and their relation to dopamine and nicotinic acetylcholine receptors in the nervous system. Neural activity involving these receptors play a vital role in complex brain functions including cognition, attention, and memory. As a result, studying them should further our understanding of drug development for the treatment of several human brain disorders.

A) Ionotropic receptor. B) Metabotropic receptor

          Before describing Nordman's research, a few background concepts should be explained. Ionotropic and metabotropic receptors vary in speed and duration of their effects. Ionotropic binds a neurotransmitter, opens the channel, allows an immediate flow of ions, and induces an EPSP or IPSP. Metabotropic binds a NT and allows a cascade of secondary messenger systems to occur. This may include but is not limited to: 1) opening another channel via an internal binding site, 2) increasing or decreasing transcription, and 3) protein modifications, including phosphorylation. Furthermore, scaffold proteins act as crucial regulators of these many signaling pathways. Their functions include opening new signaling cascades, act as new drug targets, novel net plasticity mechanisms, provide greater inter-connectivity between neurons, and even more functions we have yet to discover.

Growth cone structure

          The specific focus of Nordman's research is on the growth cone. It is a complex structure composed of three layers which guides axon development. The seven states it can be found in are initiation, formation, guidance, branching, turning, arrest, and retraction. Each growth cone state performs the function their name implies.

          Mr. Nordman and his team have found that alpha 7 nAChR (nicotinic acetylcholine receptor) are enriched within growth cones. He research also focuses on a newly discovered cytoskeletal regulator termed G protein regulated inducer of neurite outgrowth 1 (GPRIN1). GPRIN1, also enriched in growth cones, scaffolds nAChRs within neurons. This brings us to his current study which investigates how alpha 7 nAChR regulates growth cone dynamics and axon targeting in the cortex and hippocampus during early brain development. The techniques being used include subcellular fractionation to isolate growth cones from newborn pups for proteomic analysis. They observed alpha 7 nAChR/GPC in the hippocampus under a microscope by highlighting it with anibodies fluorescent, a yellow signal.

A video depicting growth cone guidance

          When in development were these proteins present? Nordman responded, the highest expression was during the guidance period. It was most abundant in the soma/GC which makes sense because that is where we expect the highest demand. How can we prove alpha 7/GPC interactions were present in the GC? Nordman stated neuron2a were neuron-like cells. He used transfection, planting DNA into a cell to produce protein; followed by immunoprecipation, removing many subunits of cells to focus on relevant network he was measuring. By eliminating GP1 from scaffold to limit interaction with GP1 and alpha 7, the weakened link suggested they are in fact connected. To confirm active apha nAChR are present in the growth cone, Nordman injected calcium sensors in the tissue grown in the petri dish. Using PNU282987, an alpha 7 activator, a green fluorescent was present which suggested alpha 7 was, in fact, in these cells.

Molecular model of
alpha 7 nicotinic receptor

          While further investigating their interactions, Nordman stated GPRIN1 is the master switch for alpha 7 signaling. During a 12 hour test, he eliminated the expression of GPRIN1 which resulted in shorter and less branching of neurons. This suggested GPRIN1 and alpha are complementary and both are vital in growth cone function. To mediate alpha 7 growth, EB3 (end binding) comets, a microtubule capping protein, was used. Using time lapse to give us real time changes of growth, EB3 moved at approximately 3-5 microns every 10 seconds.

          Mr. Nordman concluded that in "active state", filopodia projects in all directions. It commits to a direction through microtubules invasion or collapse filopodia. Alpha 7 activation inhibits G proteins mediated pathways involved in growth. The sum effect is microtubule capping and growth cone collapse.

Fewer pathways cause symptoms of schizophrenia.
This image shows neuron pathway (green strands)
comparison between a healthy mouse (left)
and one bredto express schizophrenia (right).

          Based on Nordman's findings, could it be possible to reverse this phenotype by inhibiting alpha 7 and prevent neuronal damage and promote axonal growth? He emphasized the incredibly complicated process of regenerating axons but was optimistic that it is possible. Will it have any effects on multiple sclerosis since it mainly affects myelin sheath? Perhaps an alpha 7 antagonist could be the cause of promoting axons regeneration that will effectively treat or cure schizophrenia, alzheimer's or spinal cord injuries?


Reference: http://krasnow.gmu.edu/kabbani/research-2/