|T H E N I H C A T A L Y S T||M A R C H - A P R I L 2000|
|P E O P L E|
Andres Buonanno received his Ph.D. in 1987 from Washington University Medical School in St. Louis, where he worked under the mentorship of the late John Merlie. He joined the NICHD Laboratory of Developmental Neurobiology in 1988 and is now a senior investigator heading the Section on Molecular Neurobiology.
A fundamental objective in neurobiology is to understand how connections in the nervous system are remodeled during development and by experience. Although we have known for more than two decades that electrical currents elicited by neurons have profound effects on the expression of receptors, channels, and other structural proteins, little is known of the molecular pathways that couple neural depolarization to specific changes in gene expression.
There is increasing evidence that calcium influx is an important constituent of activity-dependent regulation. But there are more questions than answers in resolving how calcium is coupled to changes in gene transcription. How are different frequencies or patterns of action potentials sensed, and then decoded, to selectively repress or stimulate transcription? Which signal transduction pathways and transcription factors are involved?
To identify the molecular pathways that mediate activity-transcription coupling in excitable tissues, my laboratory has used two experimental systems: In the brain, we have studied the activity-dependent regulation of glutamate neurotransmitter receptors; and in muscle, we have analyzed genes that are differentially regulated by specific patterns of depolarization.
The opening of an NMDA receptor by its neurotransmitter, glutamate, results in synaptic calcium currents, activation of signal transduction pathways, and a prolonged strengthening of synaptic transmission. This activity-dependent process, known as long-term potentiation, underlies complex behaviors such as learning and memory and the wiring of the nervous system. Interestingly, NMDA receptors are not only necessary to mediate these activity-dependent changes in the brain but, as we found, the subunit composition of the receptor is regulated by neural activity.
The signaling properties of NMDA receptors, which comprise a common NR1 and distinct NR2 subunits (NR2A-D), depend on the heteromeric composition of the receptor. The most striking change in the composition of the receptor occurs in the developing cerebellar granule neurons, where the expression of the NR2B subunit is shut off and replaced by expression of the NR2C subunits. We initially observed that this subunit switch, which has profound effects on the functional properties of the receptor, coincides with the innervation of granule neurons by incoming mossy fiber inputs during the first weeks of postnatal development.
We then tested the hypothesis that the innervation of granule neurons, and their depolarization by the glutamatergic mossy fiber inputs, is responsible for the NR2 subunit switch. Using transgenic mice and transfected granule neurons, we identified DNA regulatory sequences that confer neural specificity and repress the NR2B gene in response to neural activity. We then demonstrated that the upregulation of the NR2C gene requires signaling via two converging pathways: the activation of NMDA and ErbB receptors. The ErbBs are tyrosine kinase receptors that bind the neurotrophic factor neuregulin, which is found together with glutamate at the mossy fiber synapses. This was the first demonstration of factors regulating NMDA receptors and a functional role of neuregulin in neurons.
Consistent with the developmental requirement for NMDA and ErbB receptor co-signaling, we recently demonstrated that these receptors are co-localized at a structure known as the postsynaptic density (PSD). The PSD is a dense plaque rich in neurotransmitter receptors and channels adjacent to the presynaptic terminal that serves to couple trans-synaptic signaling. Using the yeast-2-hybrid system and biochemical assays, we demonstrated that the carboxyl end of the ErbB receptor interacts with the same PDZ domain-containing proteins that associate with NMDA receptors at PSDs. Proteins harboring PDZ domains, a motif necessary for protein-protein interactions, may act as "transductosomes" because they physically link membrane neurotransmitter receptors to signal-transducing enzymes in the cell.
A major emphasis of our future studies will be to analyze how activity coupled to neuregulin signaling regulates NR2 expression in other regions of the brain. Experiments are in progress to identify the factors that couple signaling via these pathways to transcription of NMDA receptor genes.
Another goal of our research is to understand how neural activity and specific depolarization frequencies regulate muscle function. The properties of slow (red) and fast (white) skeletal muscles are determined by the differential transcription of genes encoding contractile proteins. These genes are regulated by the slow or fast frequencies of motor neuron action potentials used to depolarize muscles.
We analyzed the expression of two contractile proteins known as troponins (Tn) that are selectively expressed in either slow- or fast-twitch muscles. Initially we demonstrated that Tn slow and fast expression is reversibly switched by simply changing the frequencies used to artificially depolarize denervated rat muscles. Interestingly, we found that activity can elicit opposing effects on the transcription of different genes. Transcription of Tn genes is stimulated by specific frequencies of depolarization, whereas expression of the "master regulatory factors"MyoD and myogeninis repressed by electrical activity, irrespective of frequency.
To isolate the factors that regulate the Tn genes, we began by identifying the regulatory sequences that confer specificity. Because fiber types do not develop in vitro, we used transgenic mice to identify the first enhancers known to confer either slow or fast fiber-type specificity in muscle. Surprisingly, we found that the Tn SURE (slow upstream regulatory element) and FIRE (fast intronic regulatory element) harbored four homologous DNA elements essential for activity and differed in a novel fifth element. By cutting SURE or FIRE in half and expressing the deletions in mice, we found that the downstream halves directed expression in all types of skeletal muscles (not other types of tissues), whereas the upstream regions harboring the novel element were necessary to confer slow or fast muscle specificity. The DNA element is now being used in a yeast-1-hybrid system to clone cDNAs that may encode the regulatory factors that are modulated by neural impulses and that regulate muscle fiber-type specificity.
Using these experimental approaches, we hope to understand the intriguing regulatory puzzle of how specific patterns of neural activity are sensed, and then decoded, to modify the properties of neurons and muscles in response to experience.
Mark Mattson received his Ph.D. in biology from the University of Iowa in Iowa City in 1986. After postdoctoral work at Colorado State University in Fort Collins, he took a faculty position at the Sanders-Brown Research Center on Aging at the University of Kentucky in Lexington, where he advanced to full professor in 1997. He joined NIH in 2000 as chief of the Laboratory of Neurosciences at the NIA Gerontology Research Center in Baltimore.
The long-term goal of research in my laboratory is to elucidate the molecular and cellular mechanisms responsible for nerve cell dysfunction and degeneration in age-related disorders such as Alzheimers and Parkinsons diseases. Complementary studies are aimed at identifying environmental and genetic factors that allow individuals to age successfully with little or no brain dysfunction. Our work uses a battery of cell culture and animal models of neurodegenerative disorders, in combination with analyses of brain tissue from patients with the disorders.
Over the past 12 years, we have published findings on:
the function of the b-amyloid precursor protein,
the neurotoxic mechanism of amyloid b-peptide,
mutations in presenilins and how they promote age-related synaptic dysfunction and neuronal degeneration,
the signal transduction mechanisms of neurotrophic factors and cytokines that may increase or decrease neuronal resistance to age-related disease,
the mechanisms whereby dietary restriction (which extends lifespan) benefits the aging brain.
Ongoing projects include experiments to delineate the molecular events that occur locally in synaptic terminals that mediate synaptic dysfunction and degeneration in neurodegenerative disorders. For example, we have found that biochemical cascades that mediate apoptosis can be activated in pre- and postsynaptic terminals. At these locations, apoptotic cascades can modify various synaptic regulatory systems, including glutamate receptor channels, cytoskeletal components, and mitochondrial function.
We have also identified signaling pathways that can stabilize synaptic metabolism and ion homeostasis. For example, neurotrophic factorssuch as brain-derived growth factor, basic fibroblast growth factor, and activity-dependent neurotrophic factorcan enhance synaptic glucose transport and mitochondrial function; and signaling via integrins (membrane receptors activated by specific extracellular matrix proteins) through a pathway involving Akt kinase can protect neurons against synaptically driven cell death. In order to elucidate roles for injury- and stress-responsive signaling pathways in neurological disorders, we have used gene-targeting approaches to generate mice that lack specific signaling proteins. For example, we have found that mice lacking p55 tumor necrosis factor receptor are more vulnerable to excitotoxic and ischemic brain injury than are wild-type mice; mice lacking the p50 subunit of the transcription factor NF-kB are also more vulnerable to excitotoxic injury; and mice lacking acidic sphingomy-elinase exhibit a reduced cytokine response, decreased brain injury, and improved behavioral outcome in a focal cerebral ischemia stroke model.
Telomerase is a reverse transcriptase that adds a six-base DNA repeat onto the ends of chromosomes and thereby prevents their shortening. Telomerase is linked to cell immortalization and has been touted as an anti-aging enzyme. We have found that TERT, the catalytic subunit of telomerase, is widely expressed in neurons throughout the rodent brain during embryonic and early postnatal development but is absent from neurons in the adult brain.
When we suppressed telomerase expression or function using genetic and pharmacological approaches, we found that cultured embryonic brain neurons were more vulnerable to apoptosis induced by trophic factor withdrawal and insults (such as amyloid b-peptide, glutamate, and iron) relevant to the pathogenesis of Alzheimers disease and other age-related neurodegenerative disorders. We are now in the process of generating transgenic mice that express TERT in neurons in the adult brain, with the goal of determining whether TERT will help protect neurons in animal models of neurodegenerative disorders.
Our recent findings suggest that TERT may suppress neuronal apoptosis, in part by inhibiting activity of the pro-apoptotic protein p53. In collaboration with Nigel Greig (Laboratory of Neurosciences), we have found that a chemical inhibitor of p53 is effective in protecting neurons against damage and death in experimental models of neurodegenerative disorders. We are also working to identify environmental signals, such as trophic factors and hormones that may ward off age-related neuronal degeneration.
We have recently gained insight into the mechanism whereby dietary restriction may benefit neurons in the aging brain. We found that levels of "stress proteins," including heat-shock protein-70 and glucose-regulated protein-78, as well as brain-derived neurotrophic factor and nerve growth factor, are increased in neurons in several different brain regions of mice and rats maintained on a dietary restriction regimen.
In collaboration with Don Ingram and Mark Lane (Laboratory of Neurosciences), who have shown that dietary restriction retards age-related changes in monkeys, we aim to establish whether similar molecular events occur in the brains of monkeys on a dietary restriction regimen. Based on our findings, we hope to develop strategies for preventing and treating neurodegenerative disorders of aging.
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