Knowledge interface co-design of a diabetes and metabolic syndrome initiative with and for Aboriginal people living on Ngarrindjeri country.

Objectives: This research program involves two phases to identify enablers and barriers to diabetes care for Aboriginal people on Ngarrindjeri country; and co-design a strength-based metabolic syndrome and Type 2 Diabetes (T2D) remission program with the Ngarrindjeri community. Study design: A study protocol on qualitative research. Methods: The study will recruit Aboriginal people living on Ngarrindjeri country above 18 years of age with a diagnosis of metabolic syndrome or T2D. Recruitment for phases one and two will occur through the Aboriginal Health Team at the Riverland Mallee Coorong Local Health Network. The lived experiences of T2D will be explored with 10-15 Aboriginal participants, through an Aboriginal conversational technique called 'yarning' (60-90 min) in phase 1. Elders and senior community representatives (n = 20-30) will participate in four co-design workshops (2-4 h) in phase 2. Qualitative data will be transcribed and thematically analysed (NVivo version 12). The analysis will focus on protective factors for the Cultural Determinants of Health. Ethics approval was obtained from Aboriginal Health Research Ethics Committee in South Australia (04-22-1009), and Flinders University Human Research Ethics Committee (5847). Results: This work will be used to pilot the co-designed diabetes remission trial. Outcomes will be published in peer-reviewed journals, presented at conferences, focusing on following best practice guidelines from the Australian Institute of Aboriginal and Torres Strait Islander Studies and National Health and Medical Research Council. Research translation will occur through digital posters, manuals, and infographics. Conclusions: The findings will be summarised to all Aboriginal organisations involved in this study, along with peak bodies, stakeholders, Aboriginal Services, and interested participants.

Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Functional integration of new neurons into hippocampal networks and poststroke comorbidities following neonatal stroke in mice.

Stroke in the developing brain is an important cause of chronic neurological morbidities including neurobehavioral dysfunction and epilepsy. Here, we describe a mouse model of neonatal stroke resulting from unilateral carotid ligation that results in acute seizures, long-term hyperactivity, spontaneous lateralized circling behavior, impaired cognitive function, and epilepsy. Exploration-dependent induction of the immediate early gene Arc (activity-regulated cytoskeleton associated protein) in hippocampal neurons was examined in the general population of neurons versus neurons that were generated approximately 1 week after the ischemic insult and labeled with bromodeoxyuridine. Although Arc was inducible in a network-specific manner after severe neonatal stroke, it was impaired, not only in the ipsilateral injured but also in the contralateral uninjured hippocampi when examined 6 months after the neonatal stroke. Severity of both the stroke injury and the acquired poststroke epilepsy negatively correlated with Arc induction and new neuron integration into functional circuits in the injured hippocampi.

Lithium blocks a phosphoinositide-mediated cholinergic response in hippocampal slices.

The effects of lithium on inositol phosphate metabolism may account for the therapeutic actions of lithium in affective disorder. Muscarinic stimulation of the phosphoinositide system blocks synaptic inhibitory actions of adenosine in the hippocampal slice. At therapeutic concentrations, lithium diminished this muscarinic response, whereas rubidium, which does not affect phosphoinositide metabolism, had no effect. A dampening of phosphoinositide-mediated neurotransmission may explain the normalizing effects of lithium in treating both mania and depression.

Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning.

Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.

A new model to understand the career choice and practice location decisions of medical graduates.

INTRODUCTION: Australian medical education is increasingly influenced by rural workforce policy. Therefore, understanding the influences on medical graduates' practice location and specialty choice is crucial for medical educators and medical workforce planners. The South Australian Flinders University Parallel Rural Community Curriculum (PRCC) was funded by the Australian Government to help address the rural doctor workforce shortage. The PRCC was the first community based medical education program in Australia to teach a full academic year of medicine in South Australian rural general practices. The aim of this research was to identify what factors influence the career choices of PRCC graduates. METHODS: A retrospective survey of all contactable graduates of the PRCC was undertaken. Quantitative data were analysed using SPSS 14.0 for Windows. Qualitative data were entered into NVIVO 7 software for coding, and analysed using content analysis. RESULTS: Usable data were collected from 46 of the 86 contactable graduates (53%). More than half of the respondents (54%) reported being on a rural career path. A significant relationship exists between being on a rural career pathway and making the decision prior to or during medical school (p = 0.027), and between graduates in vocational training who are on an urban career path and making a decision on career specialty after graduation from medical school (p = .004). Graduates in a general practice vocational training program are more likely to be on a rural career pathway than graduates in a specialty other than general practice (p = .003). A key influence on graduates' practice location is geographic location prior to entering medical school. Key influences on graduates choosing a rural career pathway are: having a spouse/partner with a rural background; clinical teachers and mentors; the extended rural based undergraduate learning experience; and a specialty preference for general practice. A lack of rural based internships and specialist training places is influencing both urban- and rural-origin graduates to practise in urban locations. Further analysis of graduates' career pathway choices (rural or urban) and geographic background (rural or urban) was conducted. This resulted in the development of a new model, 'The Four Qs Model'. This model consists of four quadrants derived from the variables career pathway choice (rural or urban) and geographic background (rural or urban). Clustering of consistent demographic and qualitative trends unique to each quadrant was demonstrated. The distinctive clustering that emerged from the data resulted in the quadrants being renamed 'The True Believers', 'The Convertibles' 'The Frustrated' and 'The Metro Docs'. CONCLUSIONS: The PRCC is influencing graduates to choose a rural career path. The PRCC program affirms the career preferences of rural origin students while graduates with little rural exposure prior to the PRCC report being positively influenced to pursue a rural career path. The Four Qs Model is a useful model in that it demonstrates consistent themes in the characteristics of PRCC graduates and assists understanding of why they choose a rural medical career. This could be relevant to the selection of medical students into rural medical education programs and in the construction of rural curricula. The model also offers a useful framework for further research in this field.

Selective targeting of newly synthesized Arc mRNA to active synapses requires NMDA receptor activation.

Newly synthesized Arc mRNA is selectively targeted to synapses that have experienced particular patterns of activity. Here, we demonstrate that the targeting requires NMDA receptor activation. Arc expression was induced by an electroconvulsive seizure, and the newly synthesized mRNA was then targeted to synaptic sites by activating the perforant path projections to the dentate gyrus. When micropipette electrodes containing NMDA receptor antagonists (MK801 or APV) were positioned in the dentate gyrus during the stimulation period, newly synthesized Arc mRNA was transported into dendrites but did not localize in the activated lamina; instead, the mRNA remained diffusely distributed. AMPA receptor antagonists (CNQX) blocked targeting of Arc mRNA in a small region, and mGluR antagonists (MCPG) did not affect localization. These results demonstrate that NMDA receptor activation is required for the targeting of Arc mRNA to active synapses.

L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes.

Although L-type voltage-sensitive calcium channels (VSCCs) have been well characterized electrophysiologically, their role in synaptic physiology has remained unclear. To assess their involvement in synaptic regulation of gene expression, we have examined the effects of selective VSCC antagonists on basal, synaptically mediated activation of several transcription factor genes in cultured cortical neurons. Basal expression of c-fos, jun-B, zif268, and fos-B is rapidly suppressed by exposure to L-type VSCC antagonists and increased by (-)BayK-8644, a VSCC agonist. Although VSCC antagonists block kainate-induced rises in intracellular calcium and gene expression, these agents have little effect on spontaneous electrical activity or synaptically induced calcium transients in these neurons. These findings suggest that even though L-type VSCCs contribute a relatively minor component of synaptic calcium transients, they appear to play a key role in coupling synaptic excitation to activation of transcriptional events thought to contribute to neuronal plasticity.

Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites.

Polyribosomal complexes beneath postsynaptic sites on dendrites provide a substrate for local translation of particular mRNAs, but the signals that target mRNAs to synapses remain to be defined. Here, we report that high frequency activation of the perforant path projections to the dentate gyrus causes newly synthesized mRNA for the immediate-early gene (IEG) Arc to localize selectively in activated dendritic segments. Newly synthesized Arc protein also accumulates in the portion of the dendrite that had been synaptically activated. The targeting of Arc mRNA was not disrupted by locally inhibiting protein synthesis, indicating that the signals for mRNA localization reside in the mRNA itself. This novel mechanism through which newly synthesized mRNA is precisely targeted to activated synapses is well suited to play a role in the enduring forms of activity-dependent synaptic modification that require protein synthesis.

Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids.

Prostaglandins play important and diverse roles in the CNS. The first step in prostaglandin synthesis involves enzymatic oxidation of arachidonic acid, which is catalyzed by prostaglandin H(PGH) synthase, also referred to as cyclooxygenase. We have cloned an inducible form of this enzyme from rat brain that is nearly identical to a murine, mitogen-inducible cyclooxygenase identified from fibroblasts. Our studies indicate that this gene, here termed COX-2, is expressed throughout the forebrain in discrete populations of neurons and is enriched in the cortex and hippocampus. Neuronal expression is rapidly and transiently induced by seizures or NMDA-dependent synaptic activity. No expression is detected in glia or vascular endothelial cells. Basal expression of COX-2 appears to be regulated by natural synaptic activity in the developing and adult brain. Both basal and induced expression of COX-2 are inhibited by glucocorticoids, consistent with COX-2 regulation in peripheral tissues. Our studies indicate that COX-2 expression may be important in regulating prostaglandin signaling in brain. The marked inducibility in neurons by synaptic stimuli suggests a role in activity-dependent plasticity.

Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition.

Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins. We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF). In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact. This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains. Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding. These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes.

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.

Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp.

Narp (neuronal activity-regulated pentraxin) is a secreted immediate-early gene (IEG) regulated by synaptic activity in brain. In this study, we demonstrate that Narp possesses several properties that make it likely to play a key role in excitatory synaptogenesis. Narp is shown to be selectively enriched at excitatory synapses on neurons from both the hippocampus and spinal cord. Overexpression of recombinant Narp increases the number of excitatory but not inhibitory synapses in cultured spinal neurons. In transfected HEK 293T cells, Narp interacts with itself, forming large surface clusters that coaggregate AMPA receptor subunits. Moreover, Narp-expressing HEK 293T cells can induce the aggregation of neuronal AMPA receptors. These studies support a model in which Narp functions as an extracellular aggregating factor for AMPA receptors.

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.

Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins.

Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.

Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors.

Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.

Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles.

We used fluorescent in-situ hybridization and confocal microscopy to monitor the subcellular distribution of the immediate-early gene Arc. Arc RNA appeared in discrete intranuclear foci within minutes of neuronal activation and subsequently disappeared from the nucleus and accumulated in the cytoplasm by 30 minutes. The time course of nuclear versus cytoplasmic Arc RNA accumulation was distinct, and could therefore be used to infer the activity history of individual neurons at two times. Following sequential exposure of rats to two different environments or to the same environment twice, the proportion of CA1 neurons with cytoplasmic, nuclear or overlapping Arc expression profiles matched predictions derived from ensemble neurophysiological recordings of hippocampal neuronal ensembles. Arc gene induction is thus specifically linked to neural encoding processes.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo.

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein-protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein-protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.