Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

BACKGROUND: Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown. RESULTS: Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction. CONCLUSIONS: These results identify cryptic protein interaction motifs in the cytoplasmic domain of MHCI. In so doing, they suggest that the cytoplasmic domain of MHCI could participate in previously unsuspected PDZ mediated protein-protein interactions at neuronal as well as immunological synapses.

MHC class I limits hippocampal synapse density by inhibiting neuronal insulin receptor signaling.

Proteins of the major histocompatibility complex class I (MHCI) negatively regulate synapse density in the developing vertebrate brain (Glynn et al., 2011; Elmer et al., 2013; Lee et al., 2014), but the underlying mechanisms remain largely unknown. Here we identify a novel MHCI signaling pathway that involves the inhibition of a known synapse-promoting factor, the insulin receptor. Dominant-negative insulin receptor constructs decrease synapse density in the developing Xenopus visual system (Chiu et al., 2008), and insulin receptor activation increases dendritic spine density in mouse hippocampal neurons in vitro (Lee et al., 2011). We find that genetically reducing cell surface MHCI levels increases synapse density selectively in regions of the hippocampus where insulin receptors are expressed, and occludes the neuronal insulin response by de-repressing insulin receptor signaling. Pharmacologically inhibiting insulin receptor signaling in MHCI-deficient animals rescues synapse density, identifying insulin receptor signaling as a critical mediator of the tonic inhibitory effects of endogenous MHCI on synapse number. Insulin receptors co-immunoprecipitate MHCI from hippocampal lysates, and MHCI unmasks a cytoplasmic epitope of the insulin receptor that mediates downstream signaling. These results identify an important role for an MHCI-insulin receptor signaling pathway in circuit patterning in the developing brain, and suggest that changes in MHCI expression could unexpectedly regulate neuronal insulin sensitivity in the aging and diseased brain.

Complement-mediated microglial clearance of developing retinal ganglion cell axons.

In many parts of the developing vertebrate nervous system, axons are pruned to establish mature patterns of connectivity. In this issue of Neuron, Schafer et al. (2012) show that microglia may play a role in developmental axon pruning in the thalamus by engulfing presynaptic retinal ganglion cell terminals via a C3- and CR3-dependent mechanism.

MHC class I modulates NMDA receptor function and AMPA receptor trafficking.

Proteins of the major histocompatibility complex class I (MHCI) are known for their role in immunity and have recently been implicated in long-term plasticity of excitatory synaptic transmission. However, the mechanisms by which MHCI influences synaptic plasticity remain unknown. Here we show that endogenous MHCI regulates synaptic responses mediated by NMDA-type glutamate receptors (NMDARs) in the mammalian central nervous system (CNS). The AMPA/NMDA ratio is decreased at MHCI-deficient hippocampal synapses, reflecting an increase in NMDAR-mediated currents. This enhanced NMDAR response is not associated with changes in the levels, subunit composition, or gross subcellular distribution of NMDARs. Increased NMDAR-mediated currents in MHCI-deficient neurons are associated with characteristic changes in AMPA receptor trafficking in response to NMDAR activation. Thus, endogenous MHCI tonically inhibits NMDAR function and controls downstream NMDAR-induced AMPA receptor trafficking during the expression of plasticity.

Proteolytic processing of proNGF is necessary for mature NGF regulated secretion from neurons.

Nerve growth factor mediates neuronal survival, synaptogenesis, and synaptic remodeling. We utilized primary hippocampal cultures to investigate the intrinsic motifs of proNGF that might contribute to its processing and subsequent allocation to a regulated versus constitutive secretory pathway. The addition of a carboxypeptidase E motif to proNGF did not alter the secretion of NGF. However, mutagenesis of proNGF proteolytic processing sites had significant effects on the final NGF product and its secretion. The furin recognition site (R118-S-K-R121) is essential for the proper processing of proNGF to its 13.5kDa mature product and mutating the furin site exposed an alternative processing site resulting in an intermediate NGF product of approximately 22kDa. Finally, inhibiting the processing of proNGF abolished regulated secretion of the resulting NGF product. These experiments demonstrate that hippocampal neurons harbor multiple pathways to process proNGF of which the furin consensus sequence is the preferred processing site.

HSV amplicons: neuro applications.

Strategies that employ HSV amplicon vectors in the prevention and/or amelioration of pathogenic states afflicting the central nervous system (CNS) have been extensively documented in preclinical disease models. The versatility of the HSV amplicon platform allows for the implementation of therapeutic approaches that require expression of genes exhibiting neuroprotective or neuroplastic activities, or even applications that necessitate the elaboration of antigen-specific immune responses to pathogenic proteins/structures harbored within the CNS. This discourse highlights the successes and challenges encountered using HSV amplicon vectors as tools for the dissection of neural network function and as therapeutics directed against a variety of neurologic disorders.

CNS gene therapy and a nexus of complexity: systems and biology at a crossroads.

Gene therapy is a potentially promising new treatment for neurodegenerative disorders such as Alzheimer's disease (AD), which has been difficult to treat with conventional therapeutics. Viral vector-mediated somatic gene therapy is a rapidly developing methodology for providing never before achieved capability to deliver specific genes to the CNS in a highly localized and controlled manner. With the advent and refinements of this technology one focus is directed to which genes are the most appropriate to select for specific disease indications. Nerve growth factor (NGF), a potent survival factor for critical cell populations that degenerate in AD, has been chosen already for clinical gene therapy trials in human AD patients. Much knowledge about the pathophysiological underpinnings of AD is still lacking to make clear which patients may benefit from a gene therapy approach. Moreover, a detailed understanding of sustained NGF action in the normal and diseased CNS needs to be resolved before conclusions can be drawn regarding the utility of NGF gene therapy. Systematic efforts to acquire this new knowledge should compel clinically and biologically sophisticated efforts to advance gene therapy for neurodegenerative diseases.

Cognitive fatigue during a test requiring sustained attention: a pilot study.

BACKGROUND: Fatigue is common in multiple sclerosis (MS), but difficulty quantifying fatigue severity has impeded studies of its characteristics, mechanisms, and therapeutics. Motor fatigue can be objectively measured as the decline in strength occurring during sustained contractions. Analogous declines in cognitive performance occur during tasks requiring sustained attention. OBJECTIVE: To objectively measure cognitive fatigue as a decline in performance during tests requiring sustained attention. DESIGN/METHODS: Patients with clinically stable MS (n = 20) and healthy controls (n = 21) with comparable age, gender, and education completed the Paced Auditory Serial Addition Test (PASAT) and the Digit Ordering Test (DOT) at two identical test sessions separated by 4-10 days, within a month after two practice sessions. Cognitive fatigue was quantified with two pre-specified methods for each test. The reliability of cognitive fatigue assessments was evaluated with intraclass correlation coefficients (ICCs) and construct validity was evaluated using correlations with measures of self-reported fatigue, cognition, and overall impairment/disability. RESULTS: MS patients had an average of 18.7 items correct on the first 20 items of the PASATand 17.8 correct on the last 20 items, quantified as 5.3-5.8% declines in performance using the different measurement methods (P = 0.01, rejecting the null hypothesis of zero mean decline). Although MS patients as a group demonstrated a similar decline at both sessions, ICCs were relatively low. Control patients did not demonstrate significant declines in performance during PASAT administration, but tests comparing declines in MS patients and controls did not demonstrate significant differences. Fatigue was not demonstrated using the DOT, and test-retest reliability was very poor. CONCLUSIONS: MS patients have objectively measurable cognitive fatigue during administration of the PASAT.