Natural antisense transcripts regulate the neuronal stress response and excitability

Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. Here we show that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. We demonstrate that the mRNA 3′UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-à-go-go Related Gene (hERG) potassium channel. We find that the regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, our studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity. DOI: http://dx.doi.org/10.7554/eLife.01849.00

Loss of the spectraplakin short stop activates the DLK injury response pathway in drosophila

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons

SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury

The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance

RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia

How the 22q11.2 deletion predisposes to psychiatric disease is unclear. Here, the authors examine living human neuronal cells and show that 22q11.2 regulates the expression of genes linked to autism during early development, and genes linked to schizophrenia and synaptic biology in neurons. It is unclear how the 22q11.2 deletion predisposes to psychiatric disease. To study this, we generated induced pluripotent stem cells from deletion carriers and controls and utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Here, we show that upon differentiation into neural progenitor cells, the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including autism. In excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common and rare variants. To understand how the deletion contributed to these changes, we defined the minimal protein-protein interaction network that best explains gene expression alterations. We found that many genes in 22q11.2 interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways. Our findings suggest that the 22q11.2 deletion impacts genes that may converge with psychiatric risk loci to influence disease manifestation in each deletion carrier.Peer reviewe

The Role of Cytoskeletal Stability in Regulating Synapse Development and Axonal Regeneration through the DLK Pathway

Proper network information processing relies on the intact axonal and synaptic function. In this work we investigate the mechanisms regulating the formation of the synapse and axonal response to injury. During development, neurons send out axons to connect them to their distal targets. The formation of the synapse, a point of contact between the two functional units of the nervous system, is regulated by an array of genes, many of which remain unknown. Through a large-scale RNAi screen we have identified a large number of novel genes that are involved in regulating different aspects of synapse development. By separating the genes based on their function and correlating them to the phenotype that they cause we have identified a number of genes with a shared function that are involved in a particular aspect of synapse development. Notably, genes for proteasome subunits and mitotic spindle organizers are enriched in those causing defects in synaptic apposition and NMJ stability. Genes that are involved in synapse development often also play a role in other aspects of neuronal function. We pursued the function of one particular gene that is involved in synapse development, short stop. We identify a novel allele of short stop which does not cause embryonic lethality and allows us to study the role that short stop plays in synaptic development. Mutants for short stop have a striking synapse overgrowth. This synapse phenotype is caused by the upregulation of the dual leucine zipper kinase (DLK) pathway, a MAP3K known for its role in synapse development and axon regeneration. We hypothesize that the molecular function of Short Stop as an actin-microtubule cross-linker is responsible for the DLK pathway activation in the mutant. To support this model we demonstrate that knock down of the subunits of the TCP1 complex, a chaperonin that folds actin and microtubules, also results in the upregulation of the DLK pathway. These data lead us to propose that cytoskeleton destabilization is a mechanism of DLK pathway activation. We further show that activation of this pathway in the short stop mutant is sufficient to enhance axonal response to injury. Although the DLK pathway is a key regulator of the axonal injury response, it is still unclear how this pathway is activated during injury. We demonstrate that the DLK pathway can be activated by pharmacological disruption of the cytoskeleton, which also happens during a traumatic injury. This new mechanism allows us to manipulate the DLK pathway in the intact neuron and test the sufficiency of this pathway in axonal regeneration response. We show that activating the DLK pathway via pharmacological destabilization of the cytoskeleton is sufficient to enhance axonal regeneration after injury. These data establish a new mechanism of the DLK pathway activation and that the DLK pathway is not only required but also sufficient to turn on a pro-regenerative state in an uninjured neuron

NMDA Receptor Antagonists Reveal Age-Dependent Differences in the Properties of Visual Cortical Plasticity

The suggestion that NMDA receptor (NMDAR)-dependent plasticity is subunit specific, with NR2B-types required for long-term depression (LTD) and NR2A-types critical for the induction of long-term potentiation (LTP), has generated much attention and considerable debate. By investigating the suggested subunit-specific roles of NMDARs in the mouse primary visual cortex over development, we report several important findings that clarify the roles of NMDAR subtypes in synaptic plasticity. We observed that LTD was not attenuated by application of ifenprodil, an NR2B-type antagonist, or NVP-AAM007, a less selective NR2A-type antagonist. However, we were surprised that NVP-AAM007 completely blocked adult LTP (postnatal day (P) 45–90), while only modestly affecting juvenile LTP (P21-28). To assess whether this developmental transition reflected an increasing role for NR2A-type receptors with maturity, we characterized the specificity of NVP-AAM007. We found not only that NVP-AAM007 lacks discernable subunit specificity but also that the effects of NVP-AAM077 on LTP could be mimicked using subsaturating concentrations of APV, a global NMDAR antagonist. These results indicate that the effects of NVP-AAM077 on synaptic plasticity are largely explained by nonspecific blockade of NMDARs. Moreover our findings are the first to reveal a developmental increase in the sensitivity of LTP to NMDAR antagonism. We suggest that discrepant reports describing the effect of NVP-AAM077 on LTP may be partially explained by this developmental shift in the properties of LTP. These results indicate that the degree of NMDAR activation required for LTP increases with development, providing insight into a novel underlying mechanism governing the properties of synaptic plasticity

Astrocytic cell adhesion genes linked to schizophrenia correlate with synaptic programs in neurons

The maturation of neurons and the development of synapses, although emblematic of neurons, also relies on interactions with astrocytes and other glia. Here, to study the role of glia-neuron interactions, we analyze the transcriptomes of human pluripotent stem cell (hPSC)-derived neurons, from 80 human donors, that were cultured with or without contact with glial cells. We find that the presence of astrocytes enhances synaptic gene-expression programs in neurons when in physical contact with astrocytes. These changes in neurons correlate with increased expression, in the cocultured glia, of genes that encode synaptic cell adhesion molecules. Both the neuronal and astrocyte gene-expression programs are enriched for genes associated with schizophrenia risk. Our results suggest that astrocyte-expressed genes with synaptic functions are associated with stronger expression of synaptic genetic programs in neurons, and they suggest a potential role for astrocyte-neuron interactions in schizophrenia.Peer reviewe

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Summary: Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease