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.

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.

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.

Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders.

Mutations in hundreds of genes cause neurodevelopmental disorders with abnormal motor behavior alongside cognitive deficits. Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. By direct observation and manual analysis, we characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. We recorded individual 1-day-old adult flies, with mature nervous systems and prior to the onset of aging, in small arenas. We scored behavior using open-source video-annotation software to generate continuous activity timelines, which were represented graphically and quantitatively. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1+. By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. We raise the possibility that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. We also observed significantly increased grooming in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, we propose the following proteins as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.

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.