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The Giovanni Armenise-Harvard Foundation Overview The topics covered in this session clustered at the molecular end of the neuroscience spectrum. The first presentation focused on dramatic events at the synapse, where messages are transmitted nearly instantaneously from one neuron to another. The next speaker considered how synapses are formed in the first place. The final papers addressed the molecular underpinnings of two very different, larger-scale phenomena: headaches and normal circadian rhythms.
Speed is of the essence for an excited neuron, which must quickly transmit a chemical message across the synapse to the next nerve cell in line. Speedy transmission occurs as wave after wave of vesicles loaded with neurotransmitters fuse rapidly with the membrane and release their cargo. The neuron can signal continuously because it has a reserve pool of vesicles lined up behind the ones currently releasing neurotransmitters. But even these reinforcements would not be fast enough, were it not for the nerve terminalās ability to pluck used vesicle membrane and protein from the synaptic cleft and immediately recycle it into new vesicles. This research seeks to understand how neurons recognize, sort, and repackage these vital materials. Dr. Buckley's experiments used various deletion mutants of the transferrin receptor (TfR) and chimeras of a synaptic vesicle protein (synaptobrevin) and the TfR in primary neurons to determine which organelles and molecular mechanisms channeled vesicle proteins to their proper destinations in both dendrites and axons. The proteins enter the cell via clathrin-mediated endocytosis, and then travel a recycling pathway where about half a dozen different targeting signals incorporate them into a new, functional vesicle. Synaptobrevin, which is essential for fusion, is one of the best known actors in this process. This work shows that at least two independent signals, originating in the cytoplasm, are required to target synaptobrevin to synaptic vesicles. The researchers are now exploring the possibility that the recycling pathway may have other functions as well.
In the earliest days of embryonic development, how does the tentative tip of an axon know which direction to head and when to form a synaptic connection with a second neuron? One explanation is that the neuronās cell body generates positive signals that attract the growing tip of the axon or negative ones that tell it to stop, and that the axon tip is equipped with receptors that pick up these commands. Dr. de Curtis and his colleagues have recently identified a gene called cRac1B, which is specifically expressed in the embryonic nervous system of the chicken. It belongs to the Rho family GTPases, which have been implicated in cytoskeletal reorganization during neuritogenesis. The new gene appears to have a distinct role during neural development. When cRac1B is overexpressed in primary retinal neurons it raises the number of neurites per neuron and dramatically increases their branching. In contrast, cRac1A GTPase-a closely related substance found in many different cells-does not affect neuronal growth. Furthermore, expression of either an inactive or a constitutively active form of cRac1B strikingly inhibits neuritogenesis. The cRac1B GTPase stimulates growth only in neurons; in other cell types it has the same impact as cRac1A. Detailed analysis of cRac1B proteins indicates that the carboxyterminal portion is essential for increased neuritogenesis and neurite branching. The researchers are now identifying neural regulators and/or effectors implicated in Rac action during neural development.
Genetic defects in the proteins that comprise voltage-gated channels have been linked to a number of human maladies, most of which are quite rare. For example, mutations in ?1A, the pore-forming subunit of neuronal P/Q-type calcium channels, is associated with three dominantly inherited human disorders: familial hemiplegic migraine (FHM), episodic ataxia type-2 and spinocerebellar ataxia 6. Dr. Pietrobonās lab studies four ?1A mutations that occur in about half of people with FHM, and seeks to understand how each alters calcium channels and what the clinical consequences of these changes might be. She used HEK-293 cells, transiently transfected with cDNAs encoding either wild-type or mutant human ?1A-2 and the regulatory human ?2b-? and ?2e subunits. Mutations T666M and V714A, located in the pore-lining region of domain II, decreased the number of functional calcium channels in the membrane and reduced the influx of calcium. In a minority of patches, however, mutants did not change the number or function of channels. The main effect of the mutation I1815L, in IVS6 in a position similar to V714A, was a large decrease in the number of functional calcium channels in the membrane. The mutation V714A significantly increased the probability that a channel would be open; I1815L and R192Q had similar but less dramatic effects. The mutation R192Q in IS4 increased the number of functional calcium channels in the membrane, without affecting their conductance. Future inquiries will focus on the location of mutated neurons in the brains of FHM patients, and on whether cells with fewer functional channels may die earlier than normal cells.
Endogenous, self-sustaining clocks that drive many physiologic activities and behaviors have been described in organisms ranging from fungi to humans. In mammals, the master circadian clock that drives the sleep-wake cycle and other behaviors is located in the super chiasmatic nucleus of the brain, and there are autonomous clocks in the retinas as well. Only last year the first mammalian circadian gene was identified and named Clock. Dr. Weitz suspected that the CLOCK protein would turn out to be a type of transcription factor that acts as a heterodimer, and that along with an unknown team-mate it would lead to expression of a classic circadian gene called per. About 20 possible partners were tested in his lab before one, BMAL1, was shown to be co-expressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements found adjacent to the mouse per1 gene and from an identical E-box that is associated with expression of the per gene Drosophila, suggesting a conserved regulatory mechanism. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. This is the first time that biochemical activity has been defined for a circadian clock component. Now that the researchers know that CLOCK and BMAL1 can turn on transcription of the per gene, the next question is what turns it off?
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Protein targeting in neurons and endocrine cells
