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Biomedical subjects

A M Craig

Publications and source records attributed to A M Craig.

At least 37 records · Page 2Linked to original sources
PubMed2000

Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein.

NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane. The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins. Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor). A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1). A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts. DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD. Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus. We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins.

Amino Acid Sequence↗
PubMed2000

Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of actin filaments and microtubules.

The mechanisms responsible for anchoring molecular components of postsynaptic specializations in the mammalian brain are not well understood but are presumed to involve associations with cytoskeletal elements. Here we build on previous studies of neurotransmitter receptors (Allison et al., 1998) to analyze the modes of attachment of scaffolding and signal transducing proteins of both glutamate and GABA postsynaptic sites to either the microtubule or microfilament cytoskeleton. Hippocampal pyramidal neurons in culture were treated with latrunculin A to depolymerize actin, with vincristine to depolymerize microtubules, or with Triton X-100 to extract soluble proteins. The synaptic clustering of PSD-95, a putative NMDA receptor anchoring protein and a core component of the postsynaptic density (PSD), was unaffected by actin depolymerization, microtubule depolymerization, or detergent extraction. The same was largely true for GKAP, a PSD-95-interacting protein. In contrast, the synaptic clustering of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)alpha, another core component of the PSD, was completely dependent on an intact actin cytoskeleton and was partially disrupted by detergent. Drebrin and alpha-actinin-2, actin-binding proteins concentrated in spines, were also dependent on F-actin for synaptic localization but were unaffected by detergent extraction. Surprisingly, the subcellular distributions of the inhibitory synaptic proteins GABA(A)R and gephyrin, which has a tubulin-binding motif, were unaffected by depolymerization of microtubules or actin or by detergent extraction. These studies reveal an unsuspected heterogeneity in the modes of attachment of postsynaptic proteins to the cytoskeleton and support the idea that PSD-95 and gephyrin may be core scaffolding components independent of the actin or tubulin cytoskeleton.

Actins↗
PubMed2000

Signaling between the actin cytoskeleton and the postsynaptic density of dendritic spines.

The dendritic spine may be considered a fusion of a specialized actin-based structure akin to filopodia and lamellopodia, with an excitatory postsynaptic density containing glutamate receptors and signal-transducing machinery. This specialized neuronal microdomain is the site of the majority of excitatory synaptic contacts in the mammalian brain. Regulation of spine morphology, composition, and stability are likely to contribute to long-lasting changes in synaptic efficacy. Thus, understanding the function and regulation of dendritic spines is a fundamental problem ranging from molecular through behavioral neurobiology. A complete understanding of dendritic spines will require a knowledge of all the molecular components and how these components interact. Here we wish to accomplish two goals: to catalog many of the known components of hippocampal dendritic spines and suggest how these may contribute to spine function; and to compare dendritic spines with other actin-based structures, namely lamellopodia, filopodia, microvilli, and stereocilia, to gain some insight into possible common vs. specialized mechanisms of regulation of the shape, motility, and longevity of these actin-based structures.

Actins↗
PubMed2000

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

Animals↗
PubMed2000

Understanding health technology assessment.

To make the best use of scarce national health-care resources the adoption and diffusion of new technologies is now linked to evidence of their cost- as well as clinical effectiveness. Thus, the goal of medical device manufacturers today must be to provide value for money. This article details the process of conducting this assessment.

Cost-Benefit Analysis↗
PubMed1999

Sodium cyanate alters glutathione homeostasis in rodent brain: relationship to neurodegenerative diseases in protein-deficient malnourished populations in Africa.

Sodium cyanate, a neurotoxic chemical in rodents, primates and humans, is implicated in neurodegenerative disorders in protein-deficient populations subsisting in parts of Africa on the cyanogenic plant cassava. The molecular and cellular mechanisms of cyanate neurotoxicity are not understood. This study investigates the effect of sodium cyanate on glutathione (GSH) homeostasis in rodent brain and liver in vitro and in vivo. GSH levels in mouse brain were rapidly, time- and dose-dependently decreased following intraperitoneal administration of 100, 200 or 300 mg/kg sodium cyanate. By contrast, GSH disulfide (GSSG) levels were increased and GSH/GSSG ratios were decreased in a dose-dependent manner in rat brain. Sodium cyanate depleted GSH levels in all regions of mouse brain. Brain glutathione reductase activity was dose-dependently inhibited, while glutathione peroxidase activity was not affected by sodium cyanate. The disruption of GSH homeotasis, as evidenced by reduced tissue GSH/GSSG ratios, likely results from cyanate-induced inhibition of glutathione reductase activity. The results of this study suggest that cyanate neurotoxicity, and perhaps cassava-associated neurodegenerative diseases, are mediated in part by disruption of glutathione homeostasis in neural tissue.

Africa↗
PubMed1999

Synapse formation by hippocampal neurons from agrin-deficient mice.

Agrin, a proteoglycan secreted by motoneurons, is a critical organizer of synaptic differentiation at skeletal neuromuscular junctions. Agrin is widely expressed in the nervous system so other functions seem likely, but none have been demonstrated. To test roles for agrin in interneuronal synapse formation, we studied hippocampi from mutant mice that completely lack the z+ splice form of agrin essential for neuromuscular differentiation and also exhibit severely ( approximately 90%) reduced levels of all agrin isoforms (M. Gautam et al., 1996, Cell 85, 525-535). The brains of neonatal homozygous agrin mutants were often smaller than those of heterozygous and wild-type littermates, but were morphologically and histologically indistinguishable. In particular, antibodies to pre- and postsynaptic components of glutamatergic synapses were similarly coaggregated at synaptic sites in both mutants and controls. Because mutants die at birth due to neuromuscular defects, we cultured neurons to assess later stages of synaptic maturation. In primary cultures, the agrin-deficient neurons formed MAP2-positive dendrites and tau-1-positive axons. Synaptic vesicle proteins, AMPA- and NMDA-type glutamate receptors, GABAA receptors, and the putative synapse-organizing proteins PSD-95, GKAP, and gephyrin formed numerous clusters at synaptic sites. Quantitatively, the number of SV2-labeled contacts per neuron at day 5 and the number of PSD-95 clusters per dendrite length at day 18 in culture showed no significant differences between genotypes. Furthermore, exogenous z+ agrin was unable to induce ectopic accumulation of components of central glutamatergic or GABAergic synapses as it does for neuromuscular cholinergic synapses. These results indicate that the z+ forms of agrin are dispensable for glutamatergic and GABAergic synaptic differentiation in the central nervous system.

Agrin↗
PubMed1999

Localization of the brain-specific high-affinity l-proline transporter in cultured hippocampal neurons: molecular heterogeneity of synaptic terminals.

The expression of a brain-specific, high-affinity Na+-(and Cl--)dependent l-proline transporter in subpopulations of putative glutamatergic pathways in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (Fremeau et al. , Neuron 8: 915-926, 1992). To assess further the cell-type and subcellular localization of PROT, we examined its distribution in low-density cultures of embryonic rat hippocampus. PROT immunoreactivity was detected beginning at 8 days in culture in a highly punctate pattern localizing to a subset of synaptic terminals. PROT was not detected at GABAergic terminals but was specifically localized to a subset of excitatory nerve terminals. PROT-labeled terminals showed partial apposition to AMPA-type and NMDA-type glutamate receptor clusters. Immunolabeling of isolated neurons grown in microisland cultures revealed that PROT was expressed by 60% of cultured hippocampal neurons. Individual microisland cultures were immunopositive for either PROT or glutamic acid decarboxylase, but never both. In the expressing pyramidal neurons, PROT was targeted to all presynaptic terminals. These findings indicate that PROT contributes to the molecular heterogeneity of glutamatergic terminals and suggest a novel presynaptic regulatory role for PROT in excitatory transmission at specific glutamatergic synapses.

Amino Acid Transport Systems, Neutral↗
PubMed1999

Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains.

The subcellular targeting of neurotransmitter receptors is vital in controlling polarized information flow in the brain. We show here that metabotropic glutamate receptors are differentially targeted when expressed from defective viral vectors in cultured hippocampal neurons; mGluR1a and mGluR2 are targeted to dendrites and excluded from axons, whereas mGluR7 is targeted to axons and dendrites. Chimeras and deletions revealed that axon exclusion of mGluR2 versus axon targeting of mGluR7 is mediated by their 60 amino acid C-terminal cytoplasmic domains. Addition of the mGluR7 C-terminal sequence to mGluR2 or to the unrelated somatodendritic protein telencephalin (tln) induced axon targeting, indicating dominance of the axonal signal. These mGluR sorting signals represent novel plasma membrane axon/dendrite targeting signals.

Animals↗
PubMed1999

Cost-effectiveness of prophylactic nasal mupirocin in patients undergoing peritoneal dialysis based on a randomized, placebo-controlled trial.

The study objective was to measure the benefits of elimination of nasal carriage of Staphylococcus aureus by calcium mupirocin ointment in patients undergoing continuous ambulatory peritoneal dialysis. The design was a prospective, placebo-controlled, randomized clinical trial. The subjects were 267 patients recruited from nine renal units in Belgium, France and the UK. The main outcome measures were the rate of catheter exit site infection (ESI), rates of other infections and healthcare costs from the perspective of a hospital budget-holder. The rate of ESI caused by S. aureus was significantly reduced from one in 28.1 patient months to one in 99.3 patient months (P = 0.006) and there were also non-significant trends towards lower rates of ESI caused by any organism and peritonitis caused by S. aureus. In comparison with the placebo group, patients in the mupirocin group with ESI had lower antibiotic (P = 0.02) and hospitalization costs (P = 0.065). However, overall costs of antibiotic treatment, for all infections combined, were not significantly different (P = 0.2) and total antibiotic costs (including mupirocin) were significantly higher in the mupirocin group (P = 0.001). Mupirocin prophylaxis would have been cost-neutral if the rate of ESI increased to >75% in the placebo group, or if all healthcare costs increased by 40%, or if the cost of screening was reduced from Pound Sterling 15 to Pound Sterling 3 per patient, or if the cost of mupirocin treatment was reduced from Pound Sterling 93 to Pound Sterling 40 per patient year. In conclusion, savings in healthcare costs are unlikely to be sufficiently great to offset the cost of mupirocin and screening for nasal carriage of S. aureus. The decision about whether or not to implement mupirocin should depend on a local analysis of the value of preventing ESIs caused by S. aureus.

Administration, Intranasal↗
PubMed1999

Bioactivation of cyanide to cyanate in sulfur amino acid deficiency: relevance to neurological disease in humans subsisting on cassava.

Neurological disorders have been reported from parts of Africa with protein-deficient populations and attributed to cyanide (CN-) exposure from prolonged dietary use of cassava, a cyanophoric plant. Cyanide is normally metabolized to thiocyanate (SCN-) by the sulfur-dependent enzyme rhodanese. However, in protein-deficient subjects where sulfur amino acids (SAA) are low, CN may conceivably be converted to cyanate (OCN-), which is known to cause neurodegenerative disease in humans and animals. This study investigates the fate of potassium cyanide administered orally to rats maintained for up to 4 weeks on either a balanced diet (BD) or a diet lacking the SAAs, L-cystine and L-methionine. In both groups, there was a time-dependent increase in plasma cyanate, with exponential OCN- increases in SAA-deficient rats. A strongly positive linear relationship between blood CN- and plasma OCN- concentrations was observed in these animals. These data are consistent with the hypothesis that cyanate is an important mediator of chronic cyanide neurotoxicity during protein-calorie deficiency. The potential role of thiocyanate in cassava-associated konzo is discussed in relationship to the etiology of the comparable pattern of motor-system disease (spastic paraparesis) seen in lathyrism.

Animals↗
PubMed1998

Dietary deficiency of cystine and methionine in rats alters thiol homeostasis required for cyanide detoxification.

Nutritional status is an important factor in modulating the metabolic fate of xenobiotics. Sulfur amino acid (SAA) deficiency has been proposed as a risk factor for human neurological diseases among protein-poor populations subsisting on the cyanophoric plant cassava. Female Sprague-Dawley rats were used to develop and define a model of SAA deficiency for use in future studies examining cassava-related neurotoxicity. Rats were kept in metabolic cages for 7-21 d and fed a balanced diet (BD) of known composition or a comparable diet selectively deficient in methionine and cystine (SAA-free diet). Animals fed the SAA-free diet failed to thrive, lost body weight, excreted porphyrinic materials, and showed a steep and persistent reduction of urinary inorganic sulfate. In contrast, animals on the BD gained body weight and maintained baseline output of urinary inorganic sulfate. Urinary thiocyanate excretion did not differ between groups, but plasma thiocyanate concentrations reached double that in SAA-deficient rats. Increased plasma thiocyanate suggests mobilization of sulfur amino acids from endogenous sources. Liver glutathione and blood cyanide concentrations were similar in animals on the BD and the SAA-deficient diet. In summary, a diet free of methionine and cystine results in increased retention of inorganic sulfur as thiocyanate and a near absence of inorganic sulfur excretion in urine.

Animals↗
PubMed1998

Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.

We used actin-perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the actin cytoskeleton in localizing GABAA receptors, AMPA- and NMDA-type glutamate receptors, and potential anchoring proteins at postsynaptic sites. Excitatory postsynaptic sites on dendritic spines contained a high concentration of F-actin that was resistant to cytochalasin D but could be depolymerized using the novel compound latrunculin A. Depolymerization of F-actin led to a 40% decrease in both the number of synaptic NMDA receptor (NMDAR1) clusters and the number of AMPA receptor (GluR1)-labeled spines. The nonsynaptic NMDA receptors appeared to remain clustered and to coalesce in cell bodies. alpha-Actinin-2, which binds both actin and NMDA receptors, dissociated from the receptor clusters, but PSD-95 remained associated with both the synaptic and nonsynaptic receptor clusters, consistent with a proposed cross-linking function. AMPA receptors behaved differently; on GABAergic neurons, the clusters redistributed to nonsynaptic sites, whereas on pyramidal neurons, many of the clusters appeared to disperse. Furthermore, in control neurons, AMPA receptors were detergent extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons and NMDA receptors were unextractable. GABAA receptors were not dependent on F-actin for the maintenance or synaptic localization of clusters. These results indicate fundamental differences in the mechanisms of receptor anchoring at postsynaptic sites, both regarding the anchoring of a single receptor (the AMPA receptor) in pyramidal cells versus GABAergic interneurons and regarding the anchoring of different receptors (AMPA vs NMDA receptors) at a single class of postsynaptic sites on pyramidal cell dendritic spines.

Actinin↗
PubMed1998

Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture.

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.

Actinin↗
PubMed1998

Differential regional expression and ultrastructural localization of alpha-actinin-2, a putative NMDA receptor-anchoring protein, in rat brain.

Fast chemical neurotransmission is dependent on ionotropic receptors that are concentrated and immobilized at specific postsynaptic sites. The mechanisms of receptor clustering and anchoring in neuronal synapses are poorly understood but presumably involve molecular linkage of membrane receptor proteins to the postsynaptic cytoskeleton. Recently the actin-binding protein alpha-actinin-2 was shown to bind directly to the NMDA receptor subunits NR1 and NR2B (), suggesting that alpha-actinin-2 may function to attach NMDA receptors to the actin cytoskeleton. Here we show that alpha-actinin-2 is localized specifically in glutamatergic synapses in cultured hippocampal neurons. By immunogold electron microscopy, alpha-actinin-2 is concentrated over the postsynaptic density (PSD) of numerous asymmetric synapses where it colocalizes with NR1 immunoreactivity. Thus alpha-actinin-2 is appropriately positioned at the ultrastructural level to function as a postsynaptic-anchoring protein for NMDA receptors. alpha-Actinin-2 is not, however, exclusively found at the PSD; immunogold labeling was also associated with filaments and the spine apparatus of dendritic spines and with microtubules in dendritic shafts. alpha-Actinin-2 showed marked differential regional expression in rat brain. For instance, the protein is expressed at much higher levels in dentate gyrus than in area CA1 of the hippocampus. This differential regional expression implies that glutamatergic synapses in various parts of the brain differ with respect to their alpha-actinin-2 content and thus, potentially, the extent of possible interaction between alpha-actinin-2 and the NMDA receptor.

Actinin↗