Biomedical subjects

D F Clayton

Publications and source records attributed to D F Clayton.

At least 19 recordsLinked to original sources

PubMed2001

Protein-protein interactions of alpha-synuclein in brain homogenates and transfected cells.

alpha-Synuclein is a highly conserved presynaptic protein with probable roles in normal synaptic development and plasticity as well as neurodegenerative disease, although its molecular function is not yet clear. To identify potential protein binding partners of alpha-synuclein, we performed co-immunoprecipitations using a monoclonal antibody (H3C) against its C-terminus. More than 20 detectable proteins were specifically co-immunoprecipitated from zebra finch and mouse forebrain extracts. One of these, with relative mobility of 55 kDa, was identified through microsequencing as a mixture of alpha- and beta-tubulin. Tubulin was specifically recovered from a mouse forebrain cytosolic extract by a GST/alpha-synuclein fusion protein immobilized on glutathione-Sepharose beads. In the converse experiment, alpha-synuclein bound to a column prepared from purified bovine brain tubulin immobilized upon CNBr-Sepharose. alpha-Synuclein does not appear to bind assembled microtubules, however, as alpha-synuclein did not pellet with polymerized microtubules in a standard assay for microtubule-associated proteins. Likewise, when a fusion construct of alpha-synuclein and green fluorescent protein (GFP) was expressed in African green monkey kidney epithelial (CV-1) cells, the fusion protein did not colocalize with endogenous microtubules. We conclude that alpha-synuclein may interact specifically with heterodimeric tubulin, but not microtubules, in the neuronal cytosol.

Amino Acid Sequence↗

PubMed2001

Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins.

Detergent-stable multimers of alpha-synuclein have been found specifically in the brains of patients with Parkinson's disease and other neurodegenerative diseases. Here we show that recombinant alpha-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. beta- and gamma-synucleins, as well as the Parkinson's disease-associated alpha-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family's physiological functions and may also contribute to the aggregation of alpha-synuclein observed in neurodegenerative disease.

Fatty Acids, Unsaturated↗

PubMed2001

Development of song responses in the zebra finch caudomedial neostriatum: role of genomic and electrophysiological activities.

Zebra finches first form demonstrable memories of specific songs between 25 and 35 days of age--several days after fledging from the nest. What accounts for the late onset of specific song memory formation? Here we investigated physiological development of the caudomedial neostriatum (NCM), part of the avian analogue of auditory cortex and a probable component of the system involved in song perception. Two types of physiological responses were characterized: electrophysiological (single-unit spike rate) and genomic (induction of the immediate early gene zenk, also known as zif-268, egr-1, ngfi-a, krox-24). We found that by day 20, zebra finches already have robust electrophysiological responses in NCM to song stimulation. Spike activity was greater in response to conspecific songs compared to heterospecific songs, white noise, or tones, and approximately 10% of the units showed selective responses to forward versus reversed songs. In contrast, at this age the zenk gene is expressed at a constitutively high level and undergoes no further induction in response to song presentation. At day 30, electrophysiological responses remained similar, but the zenk gene began to shift from a constitutive to an inducible pattern of expression. These results are consistent with a general role for NCM in the representation of song auditory patterns, and with a role for zenk gene expression in governing the efficiency of specific song memory storage at different ages.

Action Potentials↗

PubMed2001

Testosterone regulates alpha-synuclein mRNA in the avian song system.

Alpha-synuclein is a small, highly conserved protein in vertebrates that has been linked to several neurodegenerative diseases. The avian song control system is one of the model systems in which the protein was independently discovered. Alpha-synuclein is dynamically regulated in the song system during song learning, a process in which sex steroids play a central role. We compared alpha-synuclein mRNA expression in the brains of 12 adult male chipping sparrows (Spizella passerina) treated with either testosterone or blank s.c. implants. We saw pronounced upregulation of alpha-synuclein mRNA in, as well as an increase in the volume of, the song control nucleus area X in response to exogenous testosterone. To our knowledge this is the first report of steroid regulation of synuclein gene expression in any model system.

Animals↗

PubMed2001

Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro.

Sexual differentiation of the brain is determined in part by steroids such as estrogen, which are generally assumed to arise from the gonads. Here we show that estrogens are produced autonomously in cultured juvenile male zebra finch brain slices, and this brain-derived estrogen is both necessary and sufficient to trigger formation in vitro of a key male-specific synaptic connection in the telencephalic song control circuit. Male-like development was stimulated in female slices cultured with male slices or exposed to estrogen, and estrogen antagonists inhibited song circuit development in slices of either sex. These results reveal a new mode of sex-specific neural development, induced not by differential exposure to gonadal steroids, but rather by differential synthesis of steroids in the brain.

Animals↗

PubMed2000

Interaction of human alpha-Synuclein and Parkinson's disease variants with phospholipids. Structural analysis using site-directed mutagenesis.

alpha-Synuclein has been centrally implicated in neurodegenerative disease, and a normal function in developmental synaptic plasticity has been suggested by studies in songbirds. A variety of observations suggest the protein partitions between membrane and cytosol, a behavior apparently conferred by a conserved structural similarity to the exchangeable apolipoproteins. Here we show that the capacity to bind lipids is broadly distributed across exons 3, 4, and 5 (encoding residues 1-102). Binding to phosphatidylserine-containing vesicles requires the presence of all three exons, while binding to phosphatidic acid can be mediated by any one of the three. Consistent with a "class A2" helical binding mechanism, lipid association is disrupted by introduction of charged residues along the hydrophobic face of the predicted alpha-helix and also by biotinylation of conserved lysines (which line the interfacial region). Circular dichroism spectroscopy reveals a general correlation between the amount of lipid-induced alpha-helix content and the degree of binding to PS-containing vesicles. Two point mutations associated with Parkinson's disease have little (A30P) or no (A53T) effect on lipid binding or alpha-helicity. These results are consistent with the hypothesis that alpha-synuclein's normal functions depend on an ability to undergo a large conformational change in the presence of specific phospholipids.

Cloning, Molecular↗

PubMed2000

The genomic action potential.

Neurons compute in part by integrating, on a time scale of milliseconds, many synaptic inputs and generating a digital output-the "action potential" of classic electrophysiology. Recent discoveries indicate that neurons also perform a second, much slower, integration operating on a time scale of minutes or even hours. The output of this slower integration involves a pulse of gene expression which may be likened to the electrophysiological action potential. Its function, however, is not directed toward immediate transmission of a synaptic signal but rather toward the experience-dependent modification of the underlying synaptic circuitry. Commonly termed the "immediate early gene" (IEG) response, this phenomenon is often assumed to be a necessary component of a linear, deterministic cascade of memory consolidation. Critical review of the large literature describing the phenomenon, however, leads to an alternative model of IEG function in the brain. In this alternative, IEG activation is not directed at the consolidation of memories of a specific inducing event; instead, it sets the overall gain or efficiency of memory formation and directs it to circuits engaged by behaviorally significant contexts. The net result is a sharpening of the selectivity of memory formation, a recruitment of temporally correlated associations, and an ultimate enhancement of long-term memory retrieval.

Action Potentials↗

PubMed2000

Minimal experience required for immediate-early gene induction in zebra finch neostriatum.

We show that a single presentation of a zebra finch song, 2 s in duration, will induce an "immediate-early gene" response in the caudomedial neostriatum of zebra finches (Poephila guttata). Repetition of this stimulus 10 times is sufficient to induce a maximal increase in RNA and protein, detected 30 and 90 min later respectively. Thus very brief stimuli can set in motion a slow genomic process in the brain which takes hours to resolve. Immediate-early gene function is often considered in the context of a "feedback" model (i.e., to consolidate memories of the inducing event). However, based on the long lag observed here between initiation and full expression of the molecular response, we suggest an alternative, ethologically based, "feed-forward" model in which exposure to a novel or significant context triggers an increase in the efficiency of memory capture processes for subsequent experiences.

Animals↗

PubMed1999

Synucleins in synaptic plasticity and neurodegenerative disorders.

Synucleins are small highly conserved proteins in vertebrates, especially abundant in neurons and typically enriched at presynaptic terminals. Three genes in humans produce closely related synuclein proteins, all of which share a large amphipathic domain capable of reversible binding to lipid vesicles. Alpha synuclein has been specifically implicated in neurodegenerative disease. Two point mutations are genetically linked to familial Parkinson's disease, and alpha synuclein appears to form the major fibrillary component of Lewy bodies. Alpha synuclein also contributes to the intracellular inclusions of multiple system atrophy, and a fragment has been found in senile plaques in Alzheimer's disease. Although their normal cellular functions are unknown, several observations suggest the synucleins may serve to integrate presynaptic signaling and membrane trafficking. Alpha synuclein has been identified as a potent and selective inhibitor of phospholipase D2, which produces phosphatidic acid (to which synuclein binds) and is believed to function in the partitioning of membranes between the cell surface and intracellular stores. We outline a hypothesis whereby synuclein supports localized, experience-dependent turnover of synaptic membranes. Such a process may be important for lifelong learning and memory functions and may be especially vulnerable to disruption in aging-associated neurodegenerative diseases.

Animals↗

PubMed1998

Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.

alpha-Synuclein is a highly conserved presynaptic protein of unknown function. A mutation in the protein has been causally linked to Parkinson's disease in humans, and the normal protein is an abundant component of the intraneuronal inclusions (Lewy bodies) characteristic of the disease. alpha-Synuclein is also the precursor to an intrinsic component of extracellular plaques in Alzheimer's disease. The alpha-synuclein sequence is largely composed of degenerate 11-residue repeats reminiscent of the amphipathic alpha-helical domains of the exchangeable apolipoproteins. We hypothesized that alpha-synuclein should associate with phospholipid bilayers and that this lipid association should stabilize an alpha-helical secondary structure in the protein. We report that alpha-synuclein binds to small unilamellar phospholipid vesicles containing acidic phospholipids, but not to vesicles with a net neutral charge. We further show that the protein associates preferentially with vesicles of smaller diameter (20-25 nm) as opposed to larger (approximately 125 nm) vesicles. Lipid binding is accompanied by an increase in alpha-helicity from 3% to approximately 80%. These observations are consistent with a role in vesicle function at the presynaptic terminal.

Amino Acid Sequence↗

PubMed1998

The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease.

Synuclein proteins are produced, in vertebrates, by three genes. They share structural resemblance to apolipoproteins, but are abundant in the neuronal cytosol and present in enriched amounts at presynaptic terminals. Synucleins have been specifically implicated in three diseases:Alzheimer's (AD), Parkinson's (PD) and breast cancer. In AD, a peptide derived from alpha-synuclein forms an intrinsic component of plaque amyloid. In PD, an alpha-synuclein allele is genetically linked to several independent familial cases, and the protein appears to accumulate in Lewy bodies. In breast cancer, increased expression of gamma-synuclein correlates with disease progression. In songbirds, alpha-synuclein expression is correlated with plasticity in the developing song control system. Although the normal function of synucleins is unknown, a role in membrane plasticity seems likely.

Animals↗

PubMed1998

Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson's disease and cortical Lewy body disease contain alpha-synuclein immunoreactivity.

A mutation in the alpha-synuclein gene has recently been linked to some cases of familial Parkinson's disease (PD). We characterized the expression of this presynaptic protein in the midbrain, striatum, and temporal cortex of control, PD, and dementia with Lewy bodies (DLB) brain. Control brain showed punctate pericellular immunostaining. PD brain demonstrated alpha-synuclein immunoreactivity in nigral Lewy bodies, pale bodies and abnormal neurites. Rare neuronal soma in PD brain were immunoreactive for alpha-synuclein. DLB cases demonstrated these findings as well as alpha-synuclein immunoreactivity in cortical Lewy bodies and CA2-3 neurites. These results suggest that, even in sporadic cases, there is an early and direct role for alpha-synuclein in the pathogenesis of PD and the neuropathologically related disorder DLB.

Aged↗

PubMed1997

Response modulation in the zebra finch neostriatum: relationship to nuclear gene regulation.

The sound of birdsong activates robust gene expression in the caudomedial neostriatum (NCM) of songbirds. To assess the function of this genomic response, we analyzed the temporal and quantitative relationships between electrophysiological activity and gene induction. Single units in zebra finch NCM showed large increases in firing in response to birdsong, whereas simple auditory tones tended to inhibit firing. Most cells showed little selectivity for individual songs based on total number of spikes produced. When a novel song stimulus was repeated, the cells rapidly modulated their firing rates so that the first response to a stimulus was markedly higher than consecutive responses. Even after many repetitions of a particular song, cells continued to fire in response to that stimulus, unlike the complete "habituation" observed previously for genomic activity. The initial modulation of the response to a particular song disappeared, however, once that song was repeated for 200 trials ( approximately 34 min). These results indicate a dissociation between gross physiological activity and "immediate early" gene expression: genomic activity occurs only during a subset of electrophysiological responses. We propose a model in which nuclear responses in NCM are modulated by pathways distinct from the primary auditory inputs to NCM. This would account for the changing selectivity of the genomic response and implies an active role for the cell nucleus as an integrating agent in the physiological operation of neural circuits.

Acoustic Stimulation↗

PubMed1997

Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons.

Synelfin is a presynaptic protein of unknown function that is differentially regulated in the avian song control circuit during the critical period for song learning; in humans, it gives rise to an amyloidogenic peptide found in senile plaques of Alzheimer's disease. To gain insight into the potential involvement of synelfin in synapse development, we investigated its expression in neurons cultured from the embryonic rat hippocampus. These neurons express a variety of defined synaptic proteins, and form numerous synaptic connections after several days in culture. Synapsin I, a synaptic vesicle-associated protein, was detected within one day after the neurons were put in culture, but significant immunoreactivity for synelfin was not detected until approximately 5 days in vitro (DIV). By 3 DIV, synapsin-positive puncta (previously shown to correspond to presynaptic specializations) were detected surrounding the soma and proximal dendritic processes, whereas comparable aggregations of synelfin did not appear until several days later. By 14 DIV the punctate concentrations of synelfin and synapsin overlapped completely. Thus synelfin is expressed in these cultured neurons and eventually becomes localized to presynaptic terminals, but it is absent from these specializations when they first form. We conclude that presynaptic terminals can change in molecular composition, and that synelfin is associated with later stages in synaptic development or modulation.

Amyloid↗

PubMed1997

Synelfin regulation during the critical period for song learning in normal and isolated juvenile zebra finches.

Male zebra finches (Taeniopygia guttata) learn to sing during a critical period in adolescence. We previously described a presynaptic protein, synelfin, whose mRNA is increased early in this critical period in a brain nucleus specifically implicated in song learning, lateral MAN (lMAN). In the current study, in situ hybridization was used to map this change in gene expression to the subregion of lMAN that projects to the robust nucleus of the archistriatum (RA), the principal motor output of the telencephalic circuit that controls song production. Using confocal immunofluorescence microscopy, we detected numerous puncta of synelfin immunoreactivity that apparently represent presynaptic terminals from lMAN in the RA of young males. Synelfin immunoreactivity in RA declined abruptly between 40 and 45 days of age, a time of major synaptic reorganization in RA. This change did not occur until about 10 days after the decline in synelfin mRNA in cell bodies within lMAN, indicating a relatively slow turnover of the protein in presynaptic terminals and suggesting that some of the functional changes that occur during the critical period may arise from regulatory decisions that were initiated a week or more earlier. Depriving birds of tutoring did not halt or delay the decline of synelfin mRNA in lMAN. This change in gene expression must not be a consequence of early song learning, but may reflect an innate or programmed step in song circuit development.

Amyloid↗

PubMed1997

Localized changes in immediate-early gene regulation during sensory and motor learning in zebra finches.

A complex neural system controls birdsong learning, but its organization is not understood, nor is it known why learning only occurs during a critical period in adolescence. Here, we analyzed developmental regulation in zebra finches of zenk, an immediate-early gene (IEG) implicated in memory consolidation. Basal expression was elevated within auditory telencephalon (specifically, within the caudomedial neostriatum [NCM]) during song acquisition. Expression could be further induced by song playbacks 30 days after hatching but not at 20 days nor in juveniles reared in severe isolation. Singing itself induced zenk in song production nuclei, including Area X, even in adults. Within a compartment of the robust nucleus of the archistriatum (RA), however, this response dwindled as singing matured. These results suggest that the onset of sensory memory storage may be regulated in part at NCM, and motor plasticity may be regulated at RA.

Aging↗

PubMed1997

Role of gene regulation in song circuit development and song learning.

The songbird has emerged as an important model for study of brain-behavior relationships by virtue of its rich natural advantages and from the pioneering efforts of explorers using anatomical and behavioral approaches. Now, molecular biology is providing a new and complementary paradigm for discerning songbird brain organization and function. Here, I review the work over the last 10 years that has laid the foundation for approaching songbird biology from the molecular perspective. As a result of this work, specific hypotheses can now be framed and tested regarding the mechanisms behind song circuit formation, behavioral plasticity, and the boundaries of adaptability. Age-related changes in more than 15 molecules have been observed in the song system of juvenile zebra finches, and these changes seem to define specific phases in circuit development. In adult songbirds, ordinary song-related activities such as singing and listening cause dramatic increases in gene expression in brain areas specific to each activity. The sensitivity of gene activation is modulated as a result of experience in adulthood and also changes during juvenile song learning. These studies have provided unexpected insights into the functional organization of the song circuit and the potential role of extrinsic modulatory systems in directing and limiting plastic change in the brain. With this rich base of knowledge, and techniques of gene manipulation on the horizon, answers to old questions seem within our reach: What sets the boundaries of neural plasticity? What limits learning?

Animals↗

PubMed1996

Characterization of the precursor protein of the non-A beta component of senile plaques (NACP) in the human central nervous system.

A novel and highly conserved presynaptic protein has been independently described in rodents (synuclein/SYN-1), songbirds (synelfin), and humans (the precursor protein of the non-A beta component of senile plaques, NACP); a fragment of the latter has been detected in senile plaques in Alzheimer's disease (AD). We characterized the expression of NACP in human AD and non-AD brain. A subcellular fractionation study demonstrated that NACP was mainly localized to cytosolic fractions of human temporal cortex. NACP was also detectable in various membrane and vesicular fractions, suggesting that the protein was associated with membrane structures including synaptic vesicles. Pericellular immunostaining of the neuropil was observed in neocortical and limbic regions, supporting a synaptic localization. Senile plaques in AD brains were not immunoreactive, and confocal microscopy suggested a loss of NACP immunoreactivity in cored plaques. No difference was found in the amount of protein in AD and control frontal cortex, as measured by immunoblotting. PCR analysis showed that the full-length mRNA product was the major splice form in both AD and control human brains. Thus, despite the association of a hydrophobic fragment of NACP with senile plaques, our data suggest that the precursor itself is not a significant component of plaques and NACP synthesis is not substantially altered in AD. Nevertheless, the protein is an abundant component of synaptic regions prone to degeneration in AD, and may have a role in the expression or advancement of the disease.

Adult↗