Thorsten Trimbuch

An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration

Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimers disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7−/− fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.

A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS.

Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function

Cutting out circular RNAs Circular RNAs are widespread, but their functions have been controversial. Piwecka et al. used CRISPR-Cas9 technology to remove the locus encoding the circular RNA Cdr1as from the mouse genome. Single-cell electrophysiological measurements in excitatory neurons revealed an increase in spontaneous vesicle release from the knockout mice and depression in the synaptic response with two consecutive stimuli, indicating that Cdr1as deficiency leads to dysfunction of excitatory synaptic transmission. Small RNA sequencing of several major regions of the brain showed that expression of two microRNAs, miR-7 and miR-671, that bind to Cdr1as decreased and increased, respectively. These results, along with expression analyses, suggest that neuronal Cdr1as stabilizes or transports miR-7, which in turn represses genes that are early responders to different stimuli. Science, this issue p. eaam8526 Mice lacking a circular RNA show changes in gene expression in the brain and behavioral abnormalities. INTRODUCTION Recently, a special class of RNAs has excited researchers and triggered hundreds of now-published studies. Known as circular RNAs (circRNAs), these RNAs are produced by regular transcription from genomic DNA, but the two ends of the (usually) exonic transcripts are covalently closed, probably in most cases by noncanonical splice reactions. Most circRNAs are expressed in the cytoplasm and are unusually stable, suggesting that they may have functions that diverge from those of canonical messenger RNAs (mRNAs) or long noncoding RNAs (lncRNAs). CircRNAs tend to be weakly expressed, but there are exceptions in animal brains. For example, in the mouse brain, a few hundred circRNAs are highly expressed, often with developmentally specific expression patterns that are conserved in the human brain. We previously proposed that circRNAs may, at least sometimes, serve as regulatory RNAs. A circRNA discovered by the Kjems laboratory, CDR1as, caught our attention because it was covered with >70 binding sites for the microRNA (miRNA) miR-7. Our data suggested that CDR1as might serve to alter the free concentration of miR-7. But what really is the function of CDR1as? RATIONALE We first determined which miRNAs specifically bind Cdr1as in postmortem human and mouse brains and characterized Cdr1as expression patterns. Once we had that information, we removed Cdr1as from the mouse genome to study the molecular and behavioral consequences. RESULTS We show that Cdr1as is, in the human brain, directly and massively bound by miR-7 and miR-671. In fact, Cdr1as is one of the most common transcripts targeted by miRNAs out of all brain mRNAs or lncRNAs. The expression of miRNAs was generally unperturbed in Cdr1as knockout (KO) mice, with the exception of the two miRNAs that directly interact with Cdr1as, miR-7 and miR-671, which were respectively down-regulated and up-regulated. This perturbation was posttranscriptional, consistent with a model in which Cdr1as interacts with these miRNAs in the cytoplasm. We show that Cdr1as is highly expressed (hundreds of copies within neurons) in somas and neurites, but not in glial cells. The expression of many immediate early genes (IEGs), which are markers of neuronal activity, was consistently up-regulated in KO animals. For example, c-Fos and a few other miR-7 targets were up-regulated, suggesting that IEG up-regulation can in part be explained by miR-7 down-regulation and that Cdr1as modulates neuronal activity. Cdr1as KO mice showed a strong deficit in prepulse inhibition of the startle response, a sensorimotor gating phenotype that is impaired in several human neuropsychiatric disorders. Electrophysiological measurements indicated an increase in spontaneous vesicle release in Cdr1as KO neurons, suggesting that Cdr1as plays a role in regulating synaptic transmission. CONCLUSION Mechanistically, our data indicate that Cdr1as regulates miR-7 stability or transport in neurons, whereas miR-671 regulates Cdr1as levels. Functionally, our data suggest that Cdr1as and its direct interactions with miRNAs are important for sensorimotor gating and synaptic transmission. More generally, because the brain is an organ with exceptionally high and diverse expression of circRNAs, our data suggest the existence of a previously unknown layer of biological functions carried out by circRNAs. Cdr1as is a brain-enriched circular RNA, expressed in hundreds of copies within neurons and essential for maintaining normal brain function. Genetic ablation of the Cdr1as locus in mice led to deregulation of miR-7 and miR-671 in the brain, up-regulation of immediate early genes, synaptic malfunctions, and a deficit in prepulse inhibition of the startle reflex, a behavioral phenotype associated with neuropsychiatric disorders. Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating—a deficit in the ability to filter out unnecessary information—which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Clathrin/AP-2 Mediate Synaptic Vesicle Reformation from Endosome-like Vacuoles but Are Not Essential for Membrane Retrieval at Central Synapses

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

Should I stop or should I go? The role of complexin in neurotransmitter release

When it comes to fusion with the neuronal cell membrane, does a synaptic vesicle have a choice whether to stop or to go? Recent work suggests that complexin, a tiny protein found within the synaptic terminal, contributes to the mechanism through which this choice is made. How complexin plays this consulting part and which synaptic vesicle proteins it interacts with remain open questions. Indeed, studies in mice and flies have led to the proposal of different models of complexin function. We suggest that understanding the modular nature of complexin will help us to unpick its role in synaptic vesicle release.

Re-examining how complexin inhibits neurotransmitter release

Complexins play activating and inhibitory functions in neurotransmitter release. The complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by ‘superclamp’ and ‘poor-clamp’ mutations that enhanced or decreased the complexin-I inhibitory activity in cell–cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes. DOI: http://dx.doi.org/10.7554/eLife.02391.001

Vesicular Glutamate Transporter Expression Level Affects Synaptic Vesicle Release Probability at Hippocampal Synapses in Culture

The vesicular glutamate transporter (VGLUT) plays an essential role in synaptic transmission by filling vesicles with glutamate. At mammalian synapses, VGLUT expression level determines the amount of glutamate packaged into vesicles, and the specific paralog of VGLUT expressed affects the release probability. In this study, we investigate whether there is a link between the number of VGLUTs on vesicles and release probability. We used a combination of electrophysiology and imaging techniques in cultured mouse hippocampal neurons where the VGLUT expression level has been severely altered. We found that vesicles with drastically reduced VGLUT expression were released with a lower probability. This deficit in release could only be rescued by a functional transporter, suggesting that the transport function, and not the molecular interactions, of the protein affects vesicle release. Based on these data, we propose a novel means of presynaptic vesicle release regulation—the intravesicular glutamate fill state of the vesicle.

Role of the Transmembrane Potential in the Membrane Proton Leak

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (DeltaPsim) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high DeltaPsim (similar to DeltaPsim in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with DeltaPsim. The application of DeltaPsim up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high DeltaPsim and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA- remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease DeltaPsim more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by DeltaPsim may be key in mitochondrial respiration and metabolism.

Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose–response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.