ANCHORING of ion channels at specific subcellular sites is critical for neuronal signalling, but the mechanisms underlying channel localization and clustering are largely unknown (reviewed in ref. 1). Voltage-gated K+ channels are concentrated in various neuronal domains, including presynaptic terminals, nodes of Ranvier and dendrites, where they regulate local membrane excitability. Here we present functional and biochemical evidence that cell-surface clustering of Shaker-subfamily K+ channels is mediated by the PSD-95 family of membrane-associated putative guanylate kinases, as a result of direct binding of the carboxy-terminal cytoplasmic tails to the K+ channel subunits to two PDZ (also known as GLGF or DHR) domains in the PSD-95 protein. The ability of PDZ domains to function as independent modules for protein-protein interaction, and their presence in other junction-associated molecules (such as ZO-1 (ref. 3) and syntrophin), suggest that PDZ-domain-containing polypeptides may be widely involved in the organization of proteins at sites of membrane specialization.
Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.
Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.
Dystonia is a brain disorder characterized by sustained involuntary muscle contractions. It is typically inherited as an autosomal dominant trait with incomplete penetrance. While lacking clear degenerative neuropathology, primary dystonia is thought to involve microstructural and functional changes in neuronal circuitry. In the current study, we used magnetic resonance diffusion tensor imaging and probabilistic tractography to identify the specific circuit abnormalities that underlie clinical penetrance in carriers of genetic mutations for this disorder. This approach revealed reduced integrity of cerebellothalamocortical fiber tracts, likely developmental in origin, in both manifesting and clinically nonmanifesting dystonia mutation carriers. In these subjects, reductions in cerebellothalamic connectivity correlated with increased motor activation responses, consistent with loss of inhibition at the cortical level. Nonmanifesting mutation carriers were distinguished by an additional area of fiber tract disruption situated distally along the thalamocortical segment of the pathway, in tandem with the proximal cerebellar outflow abnormality. In individual gene carriers, clinical penetrance was determined by the difference in connectivity measured at these two sites. Overall, these findings point to a novel mechanism to explain differences in clinical expression in carriers of genes for brain disease.
Whereas total loss of Lis1 is lethal, disruption of one allele of the Lis1 gene results in brain abnormalities, indicating that developing neurons are particularly sensitive to a reduction in Lis1 dosage. Here we show that Lis1 is enriched in neurons relative to levels in other cell types, and that Lis1 interacts with the microtubule motor cytoplasmic dynein. Production of more Lis1 in non-neuronal cells increases retrograde movement of cytoplasmic dynein and leads to peripheral accumulation of microtubules. These changes may reflect neuron-like dynein behaviours induced by abundant Lis1. Lis1 deficiency produces the opposite phenotype. Our results indicate that abundance of Lis1 in neurons may stimulate specific dynein functions that function in neuronal migration and axon growth.
The synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses.
Over the past 2 decades, functional imaging techniques have become commonplace in the study of brain disease. Nevertheless, very few validated analytical methods have been developed specifically to identify and measure systems-level abnormalities in living patients. Network approaches are particularly relevant for translational research in the neurodegenerative disorders, which often involve stereotyped abnormalities in brain organization. In recent years, spatial covariance mapping, a multivariate analytical tool applied mainly to metabolic images acquired in the resting state, has provided a useful means of objectively assessing brain disorders at the network level. By quantifying network activity in individual subjects on a scan-by-scan basis, this technique makes it possible to objectively assess disease progression and the response to treatment on a system-wide basis. To illustrate the utility of network imaging in neurological research, we review recent applications of this approach in the study of Parkinson disease and related movement disorders. Novel uses of the technique are discussed, including the prediction of cognitive responses to dopaminergic therapy, evaluation of the effects of placebo treatment on network activity, assessment of preclinical disease progression, and the use of automated pattern-based algorithms to enhance diagnostic accuracy.
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