Mutually Dependent Clustering of SynDIG4/PRRT1 and AMPA Receptor Subunits GluA1 and GluA2 in Heterologous Cells and Primary Neurons.

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at synapses is a predominant mechanism for regulating synaptic strength. We identified the transmembrane protein synapse differentiation-induced gene 1 (SynDIG1; SD1) as an AMPAR interacting protein that regulates excitatory synaptic strength and AMPAR number both in vitro and in vivo. The related protein SynDIG4 (SD4; also known as PRRT1) was identified in several independent proteomic screens in complex with AMPARs, suggesting that it may function as an AMPAR auxiliary factor. Here, we show that the co-expression of SD4 with GluA1 or GluA2 homomeric AMPARs in COS cells leads to a 50 or 33% increase in the mean area of AMPAR puncta, respectively. This effect is accentuated when AMPAR puncta are stratified for co-localization with SD4, resulting in a 100 and 65% increase in GluA1 and GluA2 puncta, respectively. Chimeric proteins expressing only the membrane bound domain of SD4 co-expressed with full-length GluA1 or GluA2 recapitulated the effects of wild-type (WT) SD4. Additionally, the mean puncta area of GluA1 or GluA2 chimeras expressing the membrane and C-terminal domains increased significantly when co-localized with WT SD4. Similarly, the co-expression of GluA1 or GluA2 with SD4 results in a significant increase in the mean area of SD4 puncta co-localized with GluA1 or GluA2, respectively. Last, we observed a significant increase in the co-localization of SD4 with GluA1 after glycine induced long-term potentiation (LTP). The mean size of GluA1 puncta was significantly increased when stratified, indicating that co-localization with SD4 increases synaptic GluA1 cluster size during LTP. These data indicate mutually dependent clustering of SD4 and AMPAR subunits both in COS cells and primary hippocampal neurons, suggesting a mechanism for increased synaptic strength during chemical LTP.

SynDIG4/Prrt1 Is Required for Excitatory Synapse Development and Plasticity Underlying Cognitive Function.

Altering AMPA receptor (AMPAR) content at synapses is a key mechanism underlying the regulation of synaptic strength during learning and memory. Previous work demonstrated that SynDIG1 (synapse differentiation-induced gene 1) encodes a transmembrane AMPAR-associated protein that regulates excitatory synapse strength and number. Here we show that the related protein SynDIG4 (also known as Prrt1) modifies AMPAR gating properties in a subunit-dependent manner. Young SynDIG4 knockout (KO) mice have weaker excitatory synapses, as evaluated by immunocytochemistry and electrophysiology. Adult SynDIG4 KO mice show complete loss of tetanus-induced long-term potentiation (LTP), while mEPSC amplitude is reduced by only 25%. Furthermore, SynDIG4 KO mice exhibit deficits in two independent cognitive assays. Given that SynDIG4 colocalizes with the AMPAR subunit GluA1 at non-synaptic sites, we propose that SynDIG4 maintains a pool of extrasynaptic AMPARs necessary for synapse development and function underlying higher-order cognitive plasticity.

Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function.

UNLABELLED: Synapses are specialized contacts between neurons. Synapse differentiation-induced gene I (SynDIG1) plays a critical role during synapse development to regulate AMPA receptor (AMPAR) and PSD-95 content at excitatory synapses. Palmitoylation regulates the localization and function of many synaptic proteins, including AMPARs and PSD-95. Here we show that SynDIG1 is palmitoylated, and investigate the effects of palmitoylation on SynDIG1 stability and localization. Structural modeling of SynDIG1 suggests that the membrane-associated region forms a three-helical bundle with two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region exposed to the cytoplasm. Site-directed mutagenesis reveals that C191 and C192 are palmitoylated in heterologous cells and positively regulates dendritic targeting in neurons. Like PSD-95, activity blockade in a rat hippocampal slice culture increases SynDIG1 palmitoylation, which is consistent with our prior demonstration that SynDIG1 localization at synapses increases upon activity blockade. These data demonstrate that palmitoylation of SynDIG1 is regulated by neuronal activity, and plays a critical role in regulating its stability and subcellular localization, and thereby its function. SIGNIFICANCE STATEMENT: Palmitoylation is a reversible post-translation modification that has recently been recognized as playing a critical role in the localization and function of many synaptic proteins. Here we show that activity-dependent palmitoylation of the atypical AMPA receptor auxiliary transmembrane protein SynDIG1 regulates its stability and localization at synapses to regulate function and synaptic strength.

ß2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis.

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain. Here we identify ß2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.

As human lifespan increases, a greater fraction of the population is suffering from age-related cognitive impairments, making it important to elucidate a means to combat the effects of aging. Here we report that exposure of an aged animal to young blood can counteract and reverse pre-existing effects of brain aging at the molecular, structural, functional and cognitive level. Genome-wide microarray analysis of heterochronic parabionts--in which circulatory systems of young and aged animals are connected--identified synaptic plasticity-related transcriptional changes in the hippocampus of aged mice. Dendritic spine density of mature neurons increased and synaptic plasticity improved in the hippocampus of aged heterochronic parabionts. At the cognitive level, systemic administration of young blood plasma into aged mice improved age-related cognitive impairments in both contextual fear conditioning and spatial learning and memory. Structural and cognitive enhancements elicited by exposure to young blood are mediated, in part, by activation of the cyclic AMP response element binding protein (Creb) in the aged hippocampus. Our data indicate that exposure of aged mice to young blood late in life is capable of rejuvenating synaptic plasticity and improving cognitive function.