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The Giovanni Armenise-Harvard Foundation Session 4: Neurobiology II Overview In this session, new scientific tools begin to pick apart some of neurobiology's venerable knots. For example, Dr. Elio Raviola said in his introduction, circadian biology used to mean watching how rats behaved when the light was switched on and off. The first paper described how molecular biology can pry open the mammalian circadian clock, so that some of its gears and springs can be spread out for inspection. The second presentation examined calcium pumps, certainly among the biggest and most important membrane transporters, and asked how they might be expressed during neuronal development. The next speaker tackled a classic problem, the "inside-out" migration of newly hatched neurons to their proper places in the cerebral cortex, and identified two major actors in this journey. Finally, the concluding speaker began to lift the veil on what could prove to be the master switch for neuronal exocytosis.
Circadian clocks are endogenous oscillators that drive daily rhythms and physiology, and as such they probably represent an ancient and fundamental mechanism. In mammals, the central clock is located in the super chiasmatic nucleus of the brain, with independent clocks in each retina. Two years ago, investigators at Northwestern University identified the first mammalian circadian gene, which they called Clock. Further research showed that this gene encodes a presumptive transcription factor that is closely related to a family of proteins that mediate dimerization and bind DNA. These findings immediately reminded Dr. Weitz of what his lab had learned about the workings of the Drosophila clock, where per genes make proteins that dimerize with the product of tim(timeless). This protein pair is transported to the nucleus where it somehow shuts down the per and tim genes until the proteins disappear and the genes turn on again. These findings motivated him to look for a similar feedback loop in mice. The search for a partner for CLOCK protein turned up BMAL1, which is co-expressed with CLOCK and PER1 at known circadian clock sites in brain and retina. Additional experiments showed that CLOCK-BMAL1 heterodimers activate transcription from E-box elements, a type of transcription factor binding site, located adjacent to the mouse per1 gene, and from an identical E-box known to be important for expression of per genes in Drosophila. If the CLOCK protein was mutated, however, it joined with BMAL1 to form heterodimers that bound DNA but failed to activate transcription. According to Dr. Weitz, CLOCK-BMAL1 heterodimers drive the positive component of per transcriptional oscillations, which appear to underlie circadian rhythmicity. This is the first time that binding has been proved to activate transcription. More recently, transfection studies in mice have provided direct evidence that expression of the PER protein inhibits Per1 gene activation by CLOCK-BMAL1. Protein interaction experiments and further analysis suggest that PER binding sequesters the CLOCK-BMAL1 heterodimer in a manner that keeps the transcription factor from binding to its E-box target site.
Eucaryotic cells maintain a low intracellular concentration of free Ca2+ mainly by relying on a membrane-bound ATPase, PMCA, that serves as a high-affinity pump. Calcium is also exported from cells by a low-affinity Na+/ Ca2+ exchanger (NCX). The PMCA protein has four isoforms that vary slightly in amino acid sequence, with PMCA4 being the best studied of these. In order to learn more about the others, Dr. CarafoliÕs laboratory made monoclonal antibodies to isoforms 1-3. Using these tools, they found PMCA2 and 3 only in brain tissue, whereas isoforms 1 and 4 appear ubiquitous. In order to explore the role of PMCA isoforms 1-3 in neuron development, Dr. Carafoli and his colleagues used antibodies to track changes in these key carrier proteins in cultures of rat cerebellar granule cells (CGC). They found that PMCA isoforms 2 and 3, and a splicing variant of PMCA1 (designated as PMCA1CII) are strongly upregulated in the 3 to 5 days required for full maturation of the granule cells, whereas PMCA4 is much more rapidly downregulated. These effects occur at both the transcriptional and translational levels. Maturation of cerebellar granule cells requires the sustained influx of Ca2+ through L-type channels; when this was blocked with nifedipine the up-and-down regulation of PMCAs was abolished. At variance with the upregulation of PMCA 2, 3, and 1CII, the down-regulation of PMCA4 depends on increased levels of calcineurin, a relationship that was disrupted by immunosuppressive drugs. Dr. Carafoli also identified three isoforms of NCX at work in these cultured cells. NCX I and NCX III become slightly upregulated as the granule cells mature, whereas NCX II is strongly and rapidly down-regulated in a calcineurin-dependent way. The splicing variants of NCX1 also undergo a switch during maturation. Expression of Ca2+ transporters may change because the cells need to gain better control over the increased Ca2+ influx required for the increased gene transcription that is integral to their maturation. Future research will explore the specific properties of the individual Ca2+ carrier proteins.
Neurons are born close to the inner surface of the neural tube and migrate outward to form the layers of the mammalian cerebral cortex. In these layers, neurons are grouped according to morphology. During cortical development, successive generations of neurons migrant in an "inside-out" fashion, with the first-born brain cells settling closest to home and later cohorts traveling just past them to form the next layer. Although this pattern is well-known, the factors that guide newborn neurons into place have been a mystery. Over the past several years, Dr. Tsai has not only figured out how neurons migrate but also has uncovered connections between this phenomenon and Alzheimer's disease. Her experiments suggest that normal "inside-out" migration requires that cyclin-dependent kinase 5 work closely with p35, a regulatory protein. When she and her colleagues knocked out the p35 gene in mice, and when another lab independently knocked out the gene for cdk5, each produced mice with neurons layered "outside-in." Dr. Tsai's mice were defective but viable; the animals without cdk5 were not viable. She has since found evidence suggesting that the p35/cdk5 kinase complex facilitates "inside-out" migration by regulating actin cytoskeleton dynamics and reducing cell-cell adhesion, making it easier for freshly minted neurons to slip past the ones that have already settled in their appropriate layers. When ischemia, hydrogen peroxide, or other means were used to stress the brains of mice, the animals converted p35 to p25, a protein that Dr. Tsai believes deregulates cdk5 and has no normal developmental function. Her team found massive accumulations of p25 and cdk5 in post-mortem brain samples from Alzheimer's disease patients, especially in the neurofibrillary tangles that are one major hallmark of the disease. Abnormally phosphorylated tau protein also abounds in these tangles. Future research focuses on the role of p25/cdk5 in apoptosis, and the possibility that this might be used as a target for treating neurodegenerative disorders.
Neurosecretion is the process by which cells express and release by exocytosis both the small vesicles, which contain classical neurotransmitters, and the large dense vesicles containing mixtures of amines, ATP, proteins and peptides. Because neurons and endocrine cells acquire secretory capacity during development and retain it, this is generally regarded as a stable trait that can be lost only in case of cell de-differentiation. Some years back, Dr. Meldolesi's laboratory developed what he characterizes as "a neurosecretory cell that is incompetent for secretion." Defective clones of pheochromocytoma PC12 cells appear phenotypically normal except that they lack the dense granules typical of neurosecretory cells; functionally they have lost the ability to secrete. These rat cells lack not only secretion products but also vesicle membrane proteins, including the vSNARE VAMP2, the plasmalemma tSNAREs, which are necessary for exocytosis, and various soluble regulatory proteins. The mechanism(s) sustaining the defect appear(s) to be at least in part post-transcriptional. When defective cells are fused with normal rat PC12 or with secretory human cells, or when they are transfected with one or more normal genes, neurosecretion returns to normal levels. This implies the existence of genetic controls for exocytosis, and the nature of these mechanisms is now being investigated. Symposium Topics
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Studies on the molecular mechanism of the vertebrate circadian
clock
