Comprehensive Genomics Investigation of Neboviruses Reveals Distinct Codon Usage Patterns and Host Specificity.

Neboviruses (NeVs) from the Caliciviridae family have been linked to enteric diseases in bovines and have been detected worldwide. As viruses rely entirely on the cellular machinery of the host for replication, their ability to thrive in a specific host is greatly impacted by the specific codon usage preferences. Here, we systematically analyzed the codon usage bias in NeVs to explore the genetic and evolutionary patterns. Relative Synonymous Codon Usage and Effective Number of Codon analyses indicated a marginally lower codon usage bias in NeVs, predominantly influenced by the nucleotide compositional constraints. Nonetheless, NeVs showed a higher codon usage bias for codons containing G/C at the third codon position. The neutrality plot analysis revealed natural selection as the primary factor that shaped the codon usage bias in both the VP1 (82%) and VP2 (57%) genes of NeVs. Furthermore, the NeVs showed a highly comparable codon usage pattern to bovines, as reflected through Codon Adaptation Index and Relative Codon Deoptimization Index analyses. Notably, yak NeVs showed considerably different nucleotide compositional constraints and mutational pressure compared to bovine NeVs, which appear to be predominantly host-driven. This study sheds light on the genetic mechanism driving NeVs' adaptability, evolution, and fitness to their host species.

Modulating STDP Balance Impacts the Dendritic Mosaic.

The ability for cortical neurons to adapt their input/output characteristics and information processing capabilities ultimately relies on the interplay between synaptic plasticity, synapse location, and the nonlinear properties of the dendrite. Collectively, they shape both the strengths and spatial arrangements of convergent afferent inputs to neuronal dendrites. Recent experimental and theoretical studies support a clustered plasticity model, a view that synaptic plasticity promotes the formation of clusters or hotspots of synapses sharing similar properties. We have previously shown that spike timing-dependent plasticity (STDP) can lead to synaptic efficacies being arranged into spatially segregated clusters. This effectively partitions the dendritic tree into a tessellated imprint which we have called a dendritic mosaic. Here, using a biophysically detailed neuron model of a reconstructed layer 2/3 pyramidal cell and STDP learning, we investigated the impact of altered STDP balance on forming such a spatial organization. We show that cluster formation and extend depend on several factors, including the balance between potentiation and depression, the afferents' mean firing rate and crucially on the dendritic morphology. We find that STDP balance has an important role to play for this emergent mode of spatial organization since any imbalances lead to severe degradation- and in some case even destruction- of the mosaic. Our model suggests that, over a broad range of of STDP parameters, synaptic plasticity shapes the spatial arrangement of synapses, favoring the formation of clustered efficacy engrams.

Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo.

The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer's disease (AD) and Down's syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.

Synergistic regulation of cerebellar Purkinje neuron development by laminin epitopes and collagen on an artificial hybrid matrix construct.

The extracellular matrix (ECM) creates a dynamic environment around the cells in the developing central nervous system, providing them with the necessary biochemical and biophysical signals. Although the functions of many ECM molecules in neuronal development have been individually studied in detail, the combinatorial effects of multiple ECM components are not well characterized. Here we demonstrate that the expression of collagen and laminin-1 (lam-1) are spatially and temporally correlated during embryonic and post-natal development of the cerebellum. These changes in ECM distribution correspond to specific stages of Purkinje neuron (PC) migration, somatic monolayer formation and polarization. To clarify the respective roles of these ECM molecules on PC development, we cultured cerebellar neurons on a hybrid matrix comprised of collagen and a synthetic peptide amphiphile nanofiber bearing a potent lam-1 derived bioactive IKVAV peptide epitope. By systematically varying the concentration and ratio of collagen and the laminin epitope in the matrix, we could demonstrate a synergistic relationship between these two ECM components in controlling multiple aspects of PC maturation. An optimal ratio of collagen and IKVAV in the matrix was found to promote maximal PC survival and dendrite growth, while dendrite penetration into the matrix was enhanced by a high IKVAV to collagen ratio. In addition, the laminin epitope was found to guide PC axon development. By combining our observations in vivo and in vitro, we propose a model of PC development where the synergistic effects of collagen and lam-1 play a key role in migration, polarization and morphological maturation of PCs.

A nonlinear cable framework for bidirectional synaptic plasticity.

Finding the rules underlying how axons of cortical neurons form neural circuits and modify their corresponding synaptic strength is the still subject of intense research. Experiments have shown that internal calcium concentration, and both the precise timing and temporal order of pre and postsynaptic action potentials, are important constituents governing whether the strength of a synapse located on the dendrite is increased or decreased. In particular, previous investigations focusing on spike timing-dependent plasticity (STDP) have typically observed an asymmetric temporal window governing changes in synaptic efficacy. Such a temporal window emphasizes that if a presynaptic spike, arriving at the synaptic terminal, precedes the generation of a postsynaptic action potential, then the synapse is potentiated; however if the temporal order is reversed, then depression occurs. Furthermore, recent experimental studies have now demonstrated that the temporal window also depends on the dendritic location of the synapse. Specifically, it was shown that in distal regions of the apical dendrite, the magnitude of potentiation was smaller and the window for depression was broader, when compared to observations from the proximal region of the dendrite. To date, the underlying mechanism(s) for such a distance-dependent effect is (are) currently unknown. Here, using the ionic cable theory framework in conjunction with the standard calcium based plasticity model, we show for the first time that such distance-dependent inhomogeneities in the temporal learning window for STDP can be largely explained by both the spatial and active properties of the dendrite.

Digital expression profiling of the compartmentalized translatome of Purkinje neurons.

Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions, and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons pose a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome-the translatome-from selected subcellular domains of specific neurons, and apply it to the Purkinje cells (PCs) in the rat cerebellum. We combined microdissection, translating ribosome affinity purification (TRAP) in nontransgenic animals, and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)-associated ribosomes in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities.

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC ß-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavß subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavß and interferes with the association between Cavß and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavß via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavß and its association with the Cavα1 subunit to negatively regulate VGCC activity.

A hybrid nanofiber matrix to control the survival and maturation of brain neurons.

Scaffold design plays a crucial role in developing graft-based regenerative strategies, especially when intended to be used in a highly ordered nerve tissue. Here we describe a hybrid matrix approach, which combines the structural properties of collagen (type I) with the epitope-presenting ability of peptide amphiphile (PA) nanofibers. Self-assembly of PA and collagen molecules results in a nanofibrous scaffold with homogeneous fiber diameter of 20-30 nm, where the number of laminin epitopes IKVAV and YIGSR can be varied by changing the PA concentrations over a broad range of 0.125-2 mg/ml. Granule cells (GC) and Purkinje cells (PC), two major neuronal subtypes of cerebellar cortex, demonstrate distinct response to this change of epitope concentration. On IKVAV hybrid constructs, GC density increases three-fold compared with the control collagen substrate at a PA concentration of ≥0.25 mg/ml, while PC density reaches a maximum (five-fold vs. control) at 0.25 mg/ml of PA and rapidly decreases at higher PA concentrations. In addition, adjustment of the epitope number allowed us to achieve fine control over PC dendrite and axon growth. Due to the ability to modulate neuron survival and maturation by easy manipulation of epitope density, our design offers a versatile test bed to study the extracellular matrix (ECM) contribution in neuron development and the design of optimal neuronal scaffold biomaterials.

Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif.

BACKGROUND: The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. RESULTS: Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. CONCLUSIONS: Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

Spike timing-dependent plasticity as the origin of the formation of clustered synaptic efficacy engrams.

Synapse location, dendritic active properties and synaptic plasticity are all known to play some role in shaping the different input streams impinging onto a neuron. It remains unclear however, how the magnitude and spatial distribution of synaptic efficacies emerge from this interplay. Here, we investigate this interplay using a biophysically detailed neuron model of a reconstructed layer 2/3 pyramidal cell and spike timing-dependent plasticity (STDP). Specifically, we focus on the issue of how the efficacy of synapses contributed by different input streams are spatially represented in dendrites after STDP learning. We construct a simple feed forward network where a detailed model neuron receives synaptic inputs independently from multiple yet equally sized groups of afferent fibers with correlated activity, mimicking the spike activity from different neuronal populations encoding, for example, different sensory modalities. Interestingly, ensuing STDP learning, we observe that for all afferent groups, STDP leads to synaptic efficacies arranged into spatially segregated clusters effectively partitioning the dendritic tree. These segregated clusters possess a characteristic global organization in space, where they form a tessellation in which each group dominates mutually exclusive regions of the dendrite. Put simply, the dendritic imprint from different input streams left after STDP learning effectively forms what we term a "dendritic efficacy mosaic." Furthermore, we show how variations of the inputs and STDP rule affect such an organization. Our model suggests that STDP may be an important mechanism for creating a clustered plasticity engram, which shapes how different input streams are spatially represented in dendrite.

Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics.

An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA(A)R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA(A)Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA(A)R gamma2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA(A)R increases in parallel with neuronal activity, depending on Ca(2+) influx and calcineurin activity. These results indicate that GABA(A)R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca(2+) concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.

Red-shifted voltage-sensitive fluorescent proteins.

Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging.

Alterations in glucose metabolism induce hypothermia leading to tau hyperphosphorylation through differential inhibition of kinase and phosphatase activities: implications for Alzheimer's disease.

Alzheimer's disease (AD) brains contain neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated tau protein. Regional reductions in cerebral glucose metabolism correlating to NFT densities have been reported in AD brains. Assuming that reduced glucose metabolism might cause abnormal tau hyperphosphorylation, we induced in vivo alterations of glucose metabolism in mice by starvation or intraperitoneal injections of either insulin or deoxyglucose. We found that the treatments led to abnormal tau hyperphosphorylation with patterns resembling those in early AD brains and also resulted in hypothermia. Surprisingly, tau hyperphosphorylation could be traced down to a differential effect of low temperatures on kinase and phosphatase activities. These data indicate that abnormal tau hyperphosphorylation is associated with altered glucose metabolism through hypothermia. Our results imply that serine-threonine protein phosphatase 2A plays a major role in regulating tau phosphorylation in the adult brain and provide in vivo evidence for its crucial role in abnormal tau hyperphosphorylation in AD.

ERKs regulate PKC-dependent synaptic depression and declustering of glutamate receptors in cerebellar Purkinje cells.

Phorbol esters, such as tetradecanoylphorbol 13-acetate (TPA), have been used extensively in studies of cerebellar long-term depression (LTD), based on the hypothesis that activated protein kinase C (PKC) directly mediates alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation. Here, we show that TPA-induced depression of synaptic transmission between granule cells and Purkinje cells in culture is mediated through activation of the MEK1/2-ERK1/2 pathway. Phosphorylation of ERK1/2 induced by TPA and co-application of high potassium and glutamate was greatly attenuated by preincubating Purkinje cells with the MEK1/2 (MAPK ERK kinase 1/2) inhibitor PD98059. TPA-induced depression of synaptic transmission between granule cells and Purkinje cells was attenuated by PD98059. The MEK1/2 inhibitor also suppressed declustering of the ionotropic glutamate receptor subunit 2/3 (GluR2/3) induced by TPA and co-application of high potassium and glutamate, even though phosphorylation of Ser880 of GluR2/3 was not inhibited significantly in the presence of PD98059. These results suggest that ERK1/2 plays an essential role in TPA-induced depression via regulation of GluR2/3 declustering at the synapse.

Nitric oxide activates extracellular signal-regulated kinase 1/2 and enhances declustering of ionotropic glutamate receptor subunit 2/3 in rat cerebellar Purkinje cells.

Exogenous nitric oxide (NO) donors (S-nitroso-N-acetyl-DL-penicillamine and (E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide) and simultaneous application of high potassium and glutamate led to the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in cerebellar Purkinje cells. NO-induced ERK1/2 phosphorylation was abolished by the addition of MEK (MAPK ERK kinase) inhibitor (PD98059) or oxyhemoglobin, and was significantly reduced by the PKG (cGMP-dependent protein kinase) inhibitor, KT5823. KT5823 also reduced the peak levels of ERK1/2 activation induced by high K-glutamate or NO in Purkinje cell somata. NO-induced ERK1/2 phosphorylation remained above basal levels for at least 1 h after NO-donor removal. To address the physiological role of NO-induced ERK activation in Purkinje cells, we examined the effects of NO and 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cGMP) on PKC-dependent receptor declustering, which underlies cerebellar long-term depression. TPA (12-Ø-tetradecanoylphorbol 13-acetate), a potent activator of PKC, induced GluR2/3 receptor declustering. The 8-Br-cGMP or NO-donors enhanced GluR2/3 declustering, but did not induce it. These results suggest an important role for the NO-cGMP-PKG pathway in the activation of ERK1/2 and GluR2/3 (ionotropic glutomate receptor subunit 2/3) declustering in Purkinje cells.

New role of delta2-glutamate receptors in AMPA receptor trafficking and cerebellar function.

Previous gene knockout studies have shown that the orphan glutamate receptor delta2 (GluRdelta2) is critically involved in synaptogenesis between parallel fibers and Purkinje cells during development. However, the precise function of GluRdelta2 and whether it is functional in the mature cerebellum remain unclear. To address these issues, we developed an antibody specific for the putative ligand-binding region of GluRdelta2, and application of this antibody to cultured Purkinje cells induced AMPA receptor endocytosis, attenuated synaptic transmission and abrogated long-term depression. Moreover, injection of this antibody into the subarachnoidal supracerebellar space of adult mice caused transient cerebellar dysfunction, such as ataxic gait and poor performance in the rotorod test. These results indicate that GluRdelta2 is involved in AMPA receptor trafficking and cerebellar function in adult mice.