Mohammed Akaaboune - US grants

The main objective of this project is to better understand how neurotransmitter receptor aggregations are regulated and maintained at a synapse. Newly developed techniques for monitoring changes in the number of acetylcholine receptors at individual neuromuscular junctions in living mice will be used. This approach permits quantification of the rates of receptor addition and loss from an identified synapse over periods of minutes to months. Modifications of the activity of the neuromuscular synapse will be used to analyze the role of postsynaptic activity in modulating receptor numbers. The use of high resolution confocal microscopy fluoresce recovery after photobleaching, and two color receptor labeling, will help resolve whether newly synthesized receptors are added directly to the junction or are inserted into the perijunctional region and then migrate to the junction where they are trapped. Lastly, these experiments will examine the consequences of long-term blockade of neuromuscular transmission on the subsequent function of the neuromuscular junction. This question is clinically important because patients who have been treated with neuromuscular blocking agents for several days occasionally remain paralyzed for weeks after removing the blocking agents. The etiology of this devastating syndrome is not known but seems analogous to the effects of neuromuscular blockade observed in the preliminary work that supports this application. Because activity and inactivity induced changes in receptor numbers are thought to be instrumental in long-term potentiation and depression in the central nervous system, the studies proposed here may provide fundamental information concerning the effects of activity on synaptic strength at less accessible synapses.

DESCRIPTION (provided by applicant): The goal of this proposal is to study the dynamics (the removal, the insertion, and the mobility) of the extracellular matrix acetylcholinesterase (ACHE) in the synaptic cleft in the neuromuscular junction (NMJ) of living mice. In particular, we will study what factors that might regulate its dynamic behavior. We will focus on the role of synaptic activity, a key molecule in the extracellular basal lamina (laminin alpha 4) and in postsynaptic proteins, and the dystrophin glycoprotein complex. Our second goal is to investigate whether the metabolic stability of acetylcholine receptors (AChR) in the postsynaptic membrane is influenced by AChE function. Finally we will study changes in synaptic dynamics of both AChE and AChR at a single synapse. To address these questions we will use the NMJ as a model of synapses in vivo. The use of high-resolution confocal microscopy, in vivo fluorescence imaging assay and the photo-unbinding technique that we developed recently will help us to better understand how the densities of AChE and AChR are maintained and regulated in living synapses. By investigating such questions, we should gain a better understanding of how synaptic alterations occur in less accessible central synapses. Additionally many neuromuscular diseases have either primarily or secondarily major impact on the density of AChRs and AChEs at neuromuscular junctions such as myasthenia gravis. By understanding the regulation of these key synaptic molecules, we expect to define new approaches that might be used to develop effective therapies for these devastating diseases.

The efficiency of nerve impulse transmission to muscle cells depends on the number and density of acetylcholine receptor proteins on the muscle membrane. These receptors are localized to specialized structures known as synapses, characterized by folds in the postsynaptic membrane. Receptors are firmly held in the crest of the folds by a host of specialized scaffold proteins. The goal of this study is to investigate the dynamics of one critical component of this scaffold, rapsyn, when levels of synaptic activity are manipulated.

To address the dynamics of rapsyn at the synapse, the PI will fuse jellyfish green fluorescent protein (GFP) to rapsyn and introduce the construct into the neck of the mouse (sternomastoid muscle). This makes it possible to monitor and image a particular synapse over the course of several days in the living animal. Receptor proteins will be labeled by fluorescent bungarotoxin, a snake venom that binds with high affinity to the receptor. The PI will investigate the following questions: first, what is the lifetime of rapsyn at individual synapse in vivo. Second what is the effect of rapsyn overexpression on the metabolic stability of receptors in vivo. Finally, the PI will investigate the effect of synaptic activity on the dynamics of rapsyn at individual synapses in vivo.

The PI will promote the training of graduate, undergraduate and high school students, including those of underrepresented groups. The PI has extensive experience in training undergraduate students. In addition this proposal will enhance teaching performance and learning in the areas of neuroscience. Results will be disseminated to the scientific community through publications and by presenting findings in meetings. Finally the PI's lab will be involved in outreach to the community.

DESCRIPTION (provided by applicant): Our long term goal is to better understand how neurotransmitter receptor density is maintained and regulated at mature and developing synapses in vivo. We are using the neuromuscular junction (NMJ), an excitatory cholinergic synapse between motor neurons and muscle fibers as our model system, because of the significant impact on human health of disorders at this synapse and because it provides the most accessible model system to study the process of neurotransmitter receptor recycling in vivo. Until recently receptor recycling was thought to be an exclusive property of neurotransmitter receptors in the central nervous system, but we discovered that significant numbers of acetylcholine receptors (AChRs) are recycled back onto the postsynaptic membrane after internalization, contributing to the maintenance of the postsynaptic density of receptors at mature synapses. We will pursue two goals to assess potential roles for recycling during synaptic development and in maturity and two goals to test the roles of molecules that are strong candidates for participation in the recycling process. We have focused on the roles of alpha-dystrobrevin and alpha-syntrophin in regulating the recycling of AChR onto the postsynaptic membrane because mutations in these proteins are clearly associated with human disorders of neuromuscular transmission, and mutations of these genes in mice have been demonstrated to dramatically alter the number of AChR at mature synapses, as might be expected if receptor recycling was aberrant. Understanding the molecular basis of receptor recycling could lead to more effective intervention and therapy that could increase synaptic transmission by increasing AChR number or density in neuromuscular diseases. Furthermore, it seems likely that some aspects of receptor recycling will be common to all cells, so we expect that these results will be useful not only in understanding the development of the neuromuscular junction, but will provide insights into the long-term role of receptor recycling in synaptogenesis and synaptic plasticity at less accessible central synapses. PUBLIC HEALTH RELEVANCE: The accumulation of neurotransmitter receptors directly underneath nerve terminals is essential for healthy transmission of nerve impulses to muscle. The goal of this proposal is to help us better understand the ways by which these receptors are clustered at mature synapses and shaped during the normal development of synaptic connections and develop ways to strengthen these connections when they are weakened by disease.

DESCRIPTION (provided by applicant): The maintenance of postsynaptic acetylcholine receptors at high density clusters is critical for the effectiveness of synaptic transmission at th neuromuscular junction. Recent work from our lab has unexpectedly identified a new and important role for akap, a non-kinase anchoring protein, which is encoded within the calcium/calmodulin kinase II ¿ gene. We showed that ¿kap protects acetylcholine receptors from degradation while in the secretory pathway. In both cultured muscle and heterologous cells the protective effect of ¿kap is mediated by an ubiquitin dependent mechanism. In view of these results, we propose to study whether ¿kap can protect AChR in living mice and determine the mechanistic link between ¿kap and receptor stability. Using in vivo time-lapse imaging, quantitative fluorescence imaging, and electroporation approaches, the first specific aim will test whether the knockdown of ¿kap in muscles of living wild type mice can alter the density, number or distribution of AChR. Conversely, we will test whether the overexpression of ¿kap can rescue the drastically reduced levels of AChRs at the NMJs of ¿-syntrophin and ¿-dystrobrevin knockout mice and in surgically denervated muscles. The outcomes of these studies will provide new insight into mechanisms that can enhance the number of AChRs at synapses and will be relevant for many neuromuscular synapses where the number and density are compromised by diseases. Finally, this R21 project will establish the groundwork for future studies of how ¿kap regulates the trafficking and stability of AChR or other associated proteins at the NMJ.

Stable and efficient synaptic transmission depends largely on the maintenance of a high number/density of postsynaptic receptors at synaptic sites. At the neuromuscular junction (NMJ), the synapse between spinal motor neurons and skeletal muscle cells, the mechanisms that regulate the stability of postsynaptic nicotinic acetylcholine receptors (AChRs) over the lifetime of animals remain largely unknown. Recent studies from our lab showed that ?kap, a non-kinase muscle anchoring protein encoded within the calcium/calmodulin kinase II ? gene, plays an important role in regulating the stability of nicotinic acetylcholine receptors (AChRs) by the ubiquitin-proteasome dependent mechanism. In view of these results, we propose in the first aim to investigate the effect of ?kap knockdown on the formation, maturation, and maintenance of healthy NMJs. In the second aim we propose to investigate the effect of ?kap gain-of-function during the maturation and maintenance of compromised NMJs deficient in ?-syntrophin. In the third aim we propose to investigate the link between ?-dystrobrevin/ ?kap/ the deubiquitinating protease USP9X and AChR stability in developing and mature synapses of mice deficient in ?dbn. The outcomes of these studies will be relevant for many neuromuscular diseases where the number and density of AChRs are compromised.

Stable and efficient synaptic transmission depends largely on the maintenance of a high number/density of postsynaptic receptors at synaptic sites. At the neuromuscular junction (NMJ), the synapse between spinal motor neurons and skeletal muscle cells, the mechanisms that regulate the stability of postsynaptic nicotinic acetylcholine receptors (AChRs) over the lifetime of animals remain largely unknown. Recent studies from our lab showed that ?kap, a non-kinase muscle anchoring protein encoded within the calcium/calmodulin kinase II ? gene, plays an important role in regulating the stability of nicotinic acetylcholine receptors (AChRs) and the structural integrity of the NMJ. In view of these results, we propose in the first aim to investigate the effect of ?kap knockdown during the development of healthy neuromuscular synapses. In the second aim we propose to investigate the effect of the gain of function of ?kap on the maturation and maintenance of compromised NMJs using mice deficient in the sub- complex of the dystrophin glycoprotein complex (DGC) (?-syntrophin and ?-dystrobrevin). In the third aim we propose to investigate the molecular mechanistic link between the DGC sub- complex/?kap/ the deubiquitinating protease USP9X and the stability of AChR stability in mice deficient in ?-syntrophin/?-dystrobrevin, and USP9X. The outcomes of these studies will be relevant for many neuromuscular diseases where the number and density of AChRs are compromised.

Gastric acid secretion from parietal cells is essential for food digestion and pathogen elimination in the stomach. Dysregulation of gastric acid homeostasis underlies a spectrum of acid-related diseases, including atrophic gastritis, gastric cancer, peptic and duodenal ulcers, and gastroesophageal reflux disease (GERD); GERD alone affects at least 20% of the US population. Histamine, the primary transmitter that ?switches on? H+ secretion into the stomach lumen, acts by relocating the H+-K+-ATPase proton pump from cytoplasmic tubulovesicles (TVs) onto the apical canalicular membranes via vesicular transport and fusion. However, the mechanisms by which histamine induces the exocytosis of TVs remain unclear. Most types of regulated exocytosis, including neurotransmitter release, are Ca2+-dependent, but it remains controversial whether Ca2+ is involved in histamine-triggered TV exocytosis. Human mutations of Transient Receptor Potential Mucolipin-1 (ML1), a Ca2+-permeable channel of intracellular membranes, cause achlorhydria (low acid secretion). Using super-resolution confocal imaging, and by developing a new patch-clamp method to record directly from canalicular and tubulovesicular membranes and a new organelle-targeted Ca2+ imaging method to detect Ca2+ release from TVs, we identified ML1 as a likely histamine-triggered Ca2+ release channel in TVs. The central goal of this proposal is to investigate the roles of ML1 in TV exocytosis and to use mouse models to explore the potential clinical use of ML1 agonists and inhibitors in controlling acid secretion in vivo. Using an integrative approach with Ca2+ imaging, electrophysiology, voltage imaging, electron microscopy, small molecule channel agonists and inhibitors, and transgenic mouse models, we will test the hypothesis that histamine-cAMP-PKA signaling activate ML1 and the TV K+ channel KCNQ1 to trigger TV exocytosis and acid secretion. Aim 1 is to test the roles of ML1 and PKA in histamine-induced secretion of gastric acids. Aim 2 is to investigate the roles of KCNQ1 channels in histamine-induced gastric acid secretion. Finally, Aim 3 is to determine the roles of ML1 and KCNQ1 channels in controlling gastric acid levels in vivo. Our long-term goal of the proposed research is to lay the groundwork necessary to develop new therapeutic strategies for acid-related diseases.