Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system.

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

A Necessary Role for PKC-2 and TPA-1 in Olfactory Memory and Synaptic AMPAR Trafficking in Caenorhabditis elegans.

Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1, but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans, enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory.

Reactive Oxygen Species Modulate Activity-Dependent AMPA Receptor Transport in C. elegans.

The AMPA subtype of synaptic glutamate receptors (AMPARs) plays an essential role in cognition. Their function, numbers, and change at synapses during synaptic plasticity are tightly regulated by neuronal activity. Although we know that long-distance transport of AMPARs is essential for this regulation, we do not understand the associated regulatory mechanisms of it. Neuronal transmission is a metabolically demanding process in which ATP consumption and production are tightly coupled and regulated. Aerobic ATP synthesis unavoidably produces reactive oxygen species (ROS), such as hydrogen peroxide, which are known modulators of calcium signaling. Although a role for calcium signaling in AMPAR transport has been described, there is little understanding of the mechanisms involved and no known link to physiological ROS signaling. Here, using real-time in vivo imaging of AMPAR transport in the intact C. elegans nervous system, we demonstrate that long-distance synaptic AMPAR transport is bidirectionally regulated by calcium influx and activation of calcium/calmodulin-dependent protein kinase II. Quantification of in vivo calcium dynamics revealed that modest, physiological increases in ROS decrease calcium transients in C. elegans glutamatergic neurons. By combining genetic and pharmacological manipulation of ROS levels and calcium influx, we reveal a mechanism in which physiological increases in ROS cause a decrease in synaptic AMPAR transport and delivery by modulating activity-dependent calcium signaling. Together, our results identify a novel role for oxidant signaling in the regulation of synaptic AMPAR transport and delivery, which in turn could be critical for coupling the metabolic demands of neuronal activity with excitatory neurotransmission.SIGNIFICANCE STATEMENT Synaptic AMPARs are critical for excitatory synaptic transmission. The disruption of their synaptic localization and numbers is associated with numerous psychiatric, neurologic, and neurodegenerative conditions. However, very little is known about the regulatory mechanisms controlling transport and delivery of AMPAR to synapses. Here, we describe a novel physiological signaling mechanism in which ROS, such as hydrogen peroxide, modulate AMPAR transport by modifying activity-dependent calcium signaling. Our findings provide the first evidence in support of a mechanistic link between physiological ROS signaling, AMPAR transport, localization, and excitatory transmission. This is of fundamental and clinical significance since dysregulation of intracellular calcium and ROS signaling is implicated in aging and the pathogenesis of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease.

Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses.

Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca2+) via the mitochondrial Ca2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.

Axonal mitochondria regulate gentle touch response through control of axonal actin dynamics.

Actin in neuronal processes is both stable and dynamic. The origin & functional roles of the different pools of actin is not well understood. We find that mutants that lack mitochondria, ric-7 and mtx-2; miro-1, in neuronal processes also lack dynamic actin. Mitochondria can regulate actin dynamics upto a distance ~80 µm along the neuronal process. Absence of axonal mitochondria and dynamic actin does not markedly alter the Spectrin Membrane Periodic Skeleton (MPS) in touch receptor neurons (TRNs). Restoring mitochondria inTRNs cell autonomously restores dynamic actin in a sod-2 dependent manner. We find that dynamic actin is necessary and sufficient for the localization of gap junction proteins in the TRNs and for the C. elegans gentle touch response. We identify an in vivo mechanism by which axonal mitochondria locally facilitate actin dynamics through reactive oxygen species that we show is necessary for electrical synapses & behaviour.

Subcellular Imaging of Neuronal Calcium Handling In Vivo.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo, where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Surface acoustic wave microfluidics for repetitive and reversible temporary immobilization of C. elegans.

Caenorhabditis elegans is an important genetic model for neuroscience studies, used for analyses of how genes control connectivity, neuronal function, and behavior. To date, however, most studies of neuronal function in C. elegans are incapable of obtaining microscopy imaging with subcellular resolution and behavior analysis in the same set of animals. This constraint stems from the immobilization requirement for high-resolution imaging that is incompatible with behavioral analysis using conventional immobilization techniques. Here, we present a novel microfluidic device that uses surface acoustic waves (SAW) as a non-contact method to temporarily immobilize worms for a short period (30 seconds). We optimize the SAW based protocol for rapid switching between free-swimming and immobilized states, facilitating non-invasive analysis of swimming behavior as well as high-resolution synaptic imaging in the same animal. We find that the coupling of heat and acoustic pressure play a key role in the immobilization process. We introduce a proof-of-concept longitudinal study, illustrating that the device enables repeated imaging of fluorescently tagged synaptic receptors in command interneurons and analysis of swimming behavior in the same animals for three days. This longitudinal approach provides the first correlative analysis of synaptic glutamatergic receptors and swimming behavior in aging animals. We anticipate that this device will enable further longitudinal analysis of animal motility and subcellular morphological changes during development and aging in C. elegans.

MAPK signaling and a mobile scaffold complex regulate AMPA receptor transport to modulate synaptic strength.

Synaptic plasticity depends on rapid experience-dependent changes in the number of neurotransmitter receptors. Previously, we demonstrated that motor-mediated transport of AMPA receptors (AMPARs) to and from synapses is a critical determinant of synaptic strength. Here, we describe two convergent signaling pathways that coordinate the loading of synaptic AMPARs onto scaffolds, and scaffolds onto motors, thus providing a mechanism for experience-dependent changes in synaptic strength. We find that an evolutionarily conserved JIP-protein scaffold complex and two classes of mitogen-activated protein kinase (MAPK) proteins mediate AMPAR transport by kinesin-1 motors. Genetic analysis combined with in vivo, real-time imaging in Caenorhabditis elegans revealed that CaMKII is required for loading AMPARs onto the scaffold, and MAPK signaling is required for loading the scaffold complex onto motors. Our data support a model where CaMKII signaling and a MAPK-signaling pathway cooperate to facilitate the rapid exchange of AMPARs required for early stages of synaptic plasticity.

Regulation of neuronal excitability by reactive oxygen species and calcium signaling: Insights into brain aging.

Altered cognition and inefficient learning and memory are hallmarks of brain aging resulting from many small changes in the structure and function of neurons. One such change is a decrease in excitatory synaptic transmission mediated by glutamate and its binding to the AMPA and NMDA subtypes of glutamate receptors. Why there is decreased glutamatergic transmission in aging is not well understood. Interestingly, in aged excitatory neurons, abnormal calcium homeostasis and energy production are reliably observed. These processes have also been shown to modulate the transport and delivery of glutamate receptors to synapses. Most of these channels are translated in the cell body and must be transported to synapses by molecular motors and then transferred to the synaptic surface for proper function. Despite there being little to no research on how aging impacts these transport processes, a detailed understanding of the mechanisms regulating long-distance and local transport of these channels is coming together. Here, we review recent research on how synaptic content, specifically of glutamate receptors and voltage-gated calcium channels, is normally regulated by calcium and energy production. In addition, we discuss how that regulation may change in the aged nervous system. These advances begin to detail a mechanistic explanation in which an interplay between calcium signaling and metabolism are impacted by and, in-turn, regulate the strength of excitatory synapses.

Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response.

BACKGROUND: Alzheimer's disease (AD) is characterized by beta-amyloid (Abeta) peptide-containing plaques and tau-containing neurofibrillary tangles. By intracerebral injection of Abeta(42), both pathologies have been combined in P301L tau mutant mice. Furthermore, in cell culture, Abeta(42) induces tau aggregation. While both Abeta(42) and mutant tau cause neuronal dysfunction, their modes of action are only vaguely understood. METHODS: To determine which processes are disrupted by Abeta(42) and/or P301L mutant tau, we used transcriptomic and proteomic techniques followed by functional validation and analysis of human AD tissue. RESULTS: Our transcriptomic study in the SH-SY5Y cell culture system revealed that Abeta(42) and P301L tau expression independently affect genes controlling the cell cycle and cell proliferation. Proteomics applied to Abeta(42)-treated P301L tau-expressing SH-SY5Y cells and the amygdala of Abeta(42)-injected P301L transgenic mice revealed that a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. Among the proteins identified was valosin-containing protein (VCP), a component of the quality control system during endoplasmic reticulum stress. Mutations in VCP have recently been linked to frontotemporal dementia. CONCLUSION: Our data support the mitosis failure hypothesis that claims that aberrant cell cycle reentry of postmitotic neurons induces apoptosis. Furthermore, our data underline a role of Abeta(42) in the stress response associated with protein folding.

Functional Genomics meets neurodegenerative disorders Part I: transcriptomic and proteomic technology.

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).

Functional Genomics meets neurodegenerative disorders. Part II: application and data integration.

The transcriptomic and proteomic techniques presented in part I (Functional Genomics meets neurodegenerative disorders. Part I: transcriptomic and proteomic technology) of this back-to-back review have been applied to a range of neurodegenerative disorders, including Huntington's disease (HD), Prion diseases (PrD), Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD) and Parkinson's disease (PD). Samples have been derived either from human brain and cerebrospinal fluid, tissue culture cells or brains and spinal cord of experimental animal models. With the availability of huge data sets it will firstly be a major challenge to extract meaningful information and secondly, not to obtain contradicting results when data are collected in parallel from the same source of biological specimen using different techniques. Reliability of the data highly depends on proper normalization and validation both of which are discussed together with an outlook on developments that can be anticipated in the future and are expected to fuel the field. The new insight undoubtedly will lead to a redefinition and subdivision of disease entities based on biochemical criteria rather than the clinical presentation. This will have important implications for treatment strategies.

Neuronal Activity and CaMKII Regulate Kinesin-Mediated Transport of Synaptic AMPARs.

Excitatory glutamatergic synaptic transmission is critically dependent on maintaining an optimal number of postsynaptic AMPA receptors (AMPARs) at each synapse of a given neuron. Here, we show that presynaptic activity, postsynaptic potential, voltage-gated calcium channels (VGCCs) and UNC-43, the C. elegans homolog of CaMKII, control synaptic strength by regulating motor-driven AMPAR transport. Genetic mutations in unc-43, or spatially and temporally restricted inactivation of UNC-43/CaMKII, revealed its essential roles in the transport of AMPARs from the cell body and in the insertion and removal of synaptic AMPARs. We found that an essential target of UNC-43/CaMKII is kinesin light chain and that mouse CaMKII rescued unc-43 mutants, suggesting conservation of function. Transient expression of UNC-43/CaMKII in adults rescued the transport defects, while optogenetic stimulation of select synapses revealed CaMKII's role in activity-dependent plasticity. Our results demonstrate unanticipated, fundamentally important roles for UNC-43/CaMKII in the regulation of synaptic strength.

Kinesin-1 regulates synaptic strength by mediating the delivery, removal, and redistribution of AMPA receptors.

A primary determinant of the strength of neurotransmission is the number of AMPA-type glutamate receptors (AMPARs) at synapses. However, we still lack a mechanistic understanding of how the number of synaptic AMPARs is regulated. Here, we show that UNC-116, the C. elegans homolog of vertebrate kinesin-1 heavy chain (KIF5), modifies synaptic strength by mediating the rapid delivery, removal, and redistribution of synaptic AMPARs. Furthermore, by studying the real-time transport of C. elegans AMPAR subunits in vivo, we demonstrate that although homomeric GLR-1 AMPARs can diffuse to and accumulate at synapses in unc-116 mutants, glutamate-gated currents are diminished because heteromeric GLR-1/GLR-2 receptors do not reach synapses in the absence of UNC-116/KIF5-mediated transport. Our data support a model in which ongoing motor-driven delivery and removal of AMPARs controls not only the number but also the composition of synaptic AMPARs, and thus the strength of synaptic transmission.

Cornichons control ER export of AMPA receptors to regulate synaptic excitability.

The strength of synaptic communication at central synapses depends on the number of ionotropic glutamate receptors, particularly the class gated by the agonist AMPA (AMPARs). Cornichon proteins, evolutionarily conserved endoplasmic reticulum cargo adaptors, modify the properties of vertebrate AMPARs when coexpressed in heterologous cells. However, the contribution of cornichons to behavior and in vivo nervous system function has yet to be determined. Here, we take a genetic approach to these questions by studying CNI-1--the sole cornichon homolog in C. elegans. cni-1 mutants hyperreverse, a phenotype associated with increased glutamatergic synaptic transmission. Consistent with this behavior, we find larger glutamate-gated currents in cni-1 mutants with a corresponding increase in AMPAR number. Furthermore, we observe opposite phenotypes in transgenic worms that overexpress CNI-1 or vertebrate homologs. In reconstitution studies, we provide support for an evolutionarily conserved role for cornichons in regulating the export of vertebrate and invertebrate AMPARs.

The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

A conserved function of C. elegans CASY-1 calsyntenin in associative learning.

BACKGROUND: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance. However, such studies do not allow for a functional or causal testing of newly identified candidate genes. Since polymorphisms in Calsyntenin 2 (CLSTN2) showed a significant association with episodic memory performance in humans, we tested the C. elegans CLSTN2 ortholog CASY-1 for possible functions in the associative behavior of C. elegans. METHODOLOGY/PRINCIPAL FINDINGS: Using three different associative learning paradigms and functional rescue experiments, we show that CASY-1 plays an important role during associative learning in C. elegans. Furthermore, neuronal expression of human CLSTN2 in C. elegans rescues the learning defects of casy-1 mutants. Finally, genetic interaction studies and neuron-specific expression experiments suggest that CASY-1 may regulate AMPA-like GLR-1 glutamate receptor signaling. CONCLUSION/SIGNIFICANCE: Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.