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The Giovanni Armenise-Harvard Foundation Session 3: Activities of Nerve and Muscle Overview Neuroscience is one of the broadest contemporary scientific disciplines, both in terms of its methods and what they are used to study. It encompasses everything from basic biophysics to clinical surveys aimed at linking human diseases with genetic abnormalities. Although three of the four reports in this session use molecular tools to study nerves and muscles, they nevertheless illustrate some of the field's diversity. In his introductory remarks, Dr. Elio Raviola of HMS noted that most scientific lectures about signal transduction and other intracellular events are accompanied by slides showing circles, squares, connecting lines, and directional arrows. But none of these can be seen under the microscope, he observed to appreciative laughter. In fact, little is really know about where many molecular events actually take place within living cells. The lead paper in this session described a technique that can be used to track specific chemical changes in real time. The next two presentations took a molecular look at the interface between nerves and muscles. The first considered calcium's role in the flow of information from the pre-synaptic to the post-synaptic neuron, and how this influences the activity of the synaptic cell. The second examined signal transduction pathways that link electrical signals at the muscle cell surface to transcriptional commands in its nucleus, and along the way uncovers a new role for Ras. The narrative sweep of the final paper was unusually broad. Here the researchers had to function as social scientists in the field, making contact with members of a sprawling Italian family, before they could use the tools of molecular genetics in the laboratory. The reward was the discovery of a new mutation responsible for an unusual form of epilepsy. Presentations
Although tyrosine kinase receptor cascades are important, they are only one part of the signal transduction story. Other important players include small molecules known as intracellular mediators or second messengers, especially Ca2+ and cAMP. These widely used messengers receive signals from surface receptors, and transmit signals to cellular proteins. Dr. Pozzan's lab has developed novel techniques for pinpointing second messenger activity and monitoring their dynamic interactions. His team used several fluorescent proteins, produced by the jelly fish Aequorea victoria, to engineer specialized sensors that track the activities of Ca2+ or cAMP in living cells. Some of their findings challenge the conventional wisdom. For example, the mitrochondria have traditionally been seen as the main organelle involved in Ca2+ handling. A sensor made with the Ca2+ sensitive photoprotein aequorin, which lights up and "freezes" Ca2+ activity in stimulated cells, made it possible to localize calcium activity in subpopulations of organelles. Studies with this luminescent probe revealed that in fact a subpopulation of mitochondria, which huddle around the endoplasmic reticulum and do not stray, carry out most of the calcium exchange. The researchers invented a second sensor that could be used to localize cAMP signaling. This probe tags the regulatory (RII) and catalytic (Cat) subunits of protein kinase A (PKA) with either of two types of green fluorescent protein. The two GFPs were selected for their ability to generate fluorescence resonance energy transfer, or FRET. PKA is the main effector of cAMP in eukaryotic cells. When cAMP is low inside the cell, the RII and Cat subunits of PKA are in close proximity and the donor GFP can transfer energy to the nearby acceptor GFP. When cAMP levels increase, cAMP binds to the RII subunit and the active Cat subunit is released. FRET disappears as soon as the two are separated. By measuring the ratio of blue to green emissions, Dr. Pozzan's team can localize and measure cAMP fluctuations in response to selective stimulation of plasma membrane receptors.
During the normal operation of the brain, neurons often fire in intense, high frequency bursts, separated by long silent intervals. Calcium channels open along the axon as signals travel toward the bouton where neurotransmitter is squirted into the synapse. Although synapses are known to undergo profound strength changes in response to activity, and although changing levels of calcium ions are thought to regulate the strength of the neurotransmitter message, this calcium activity has been quite difficult to observe. Several years ago, Dr. Regehr and his colleagues developed a method for using calcium-binding fluorescent dyes to study how calcium ions govern neurotransmitter release in different types of neurons. Recently, the researchers have focused on "climbing fiber" synapses that drive the Purkinje cells in rat cerebellum. Some of their findings are surprising: they expected a cell that released a huge burst of neurotransmitter to recover more slowly than a cell that delivered a smaller amount. When presynaptic cells were rapidly and repeatedly stimulated, however, they recovered much more quickly than the researchers predicted. In general, the higher the calcium level, the faster they recuperated and fired again. When the researchers experimentally manipulated calcium levels in presynaptic cells, they found that both release and recovery could be altered. Although there is still much to learn about the dynamic regulation of synaptic strength, Dr. Regehr's interpretation is that the synapses have a complex system for filtering inputs and controlling synaptic outputs during complex activity patterns.
The type of nervous stimulation that a muscle receives is an established factor in both its growth and the type of fiber it comprises. Less clear are the signal transduction pathways that link depolarization at the muscle cell's surface with transcriptional changes in its nucleus. In order to identify these pathways, Dr. Schiaffino's lab uses the rat soleus muscle, in the animal's lower limb, as an in vivo muscle regeneration model. As a result of these studies, they have found a new role for the familiar Ras signaling pathway. Local changes at the neuromuscular junction and broader changes in muscle phenotype occur when a muscle is deprived of nerve stimulation. If an injured rat soleus muscle is not renervated with several days, genes that produce fast-fiber myocin will quickly predominate. New connections will appear, but normal activity will not be restored. If natural healing takes place and the muscle is renervated, large quantities of slow-fiber myocin will be produced and function will return. In the laboratory, this effect can be reproduced by electrostimulation using a continuous, low-frequency pattern. Knowing that electrical stimulation could restore normal myocin production, Dr. Schiaffino and his colleagues manipulated the Ras signal transduction pathway. They transfected regenerating muscles with either constitutively active Ras or a negative Ras mutant. An unexpected finding was that the active Ras mutant stepped up production of slow-fiber myocin and down-regulated fast myocin - even in cells that were denervated. The dominant negative Ras, in contrast, interfered with regeneration even in electrically stimulated muscles. Additional experiments showed that selective activation of different pathways downstream of Ras had differing effects on muscle growth and fiber type. RasV12S35, which activates the MAPK (ERK) pathway, was able to induce slow myocin but not muscle growth; RasV12C40, which activates the PI3K pathway, affected muscle growth but not myosin gene expression. In addition to the traditional association of Ras and inhibition of myoblast fusion and muscle cell differentiation in culture, this study identifies a new role for this pathway in the differentiation of muscle phenotype by nerve activity.
Epilepsy is a label applied to a broad spectrum of seizure disorders, ranging from mild and occasional to frequent and severe, that affects as many as 1 in 200 people in the general population. Partial or focal forms account for about 60% of epilepsy cases, and there is evidence that a few percent of these are due to single gene abnormalities. Two gene loci have been linked with a Mendelian form of partial epilepsy, called autosomal dominant nocturnal frontal lobe epilepsy, or ADNFLE. The typical clinical presentation involves seizures during light sleep, which are frequently misdiagnosed as nightmares. Among affected members of the same pedigree, some rarely experience episodes and others are troubled by frequent, even nightly seizures. Symptoms of ANDFLE, which typically begin in childhood and do not worsen over time, usually respond well to treatment with carbamazepine or other anti-seizure medications. Dr. Casari's team learned of a large Italian family that included many members who, beginning around age 9 to 12, experienced seizures during sleep. Most had a prodrome that involved auras, shivering, or tingling sensations. The researchers used genome-wide linkage mapping to track a third locus for ADNFLE, which they call ENFL3, to a large region of chromosome 1. This region turned out to include CHRNB2, which encodes the beta-2 subunit of the neuronal nicotinic receptor (nAChR). The researchers found that family members with ADNFLE had a mis-sense mutation in this gene that resulted in this gated channel remaining open for an abnormally long time. This finding demonstrates that ADNFLE is more genetically heterogeneous than previously thought, and indicates that the cholinergic system plays a pathogenic role in this form of partial epilepsy.
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Imaging
signal transduction in living cells
