miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS.

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease\u27s etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalize

The Huntington's disease mutation impairs Huntingtin's role in the transport of NF-κB from the synapse to the nucleus

Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here, we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics, but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified 7 mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct velocity requirements

BDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons

Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation

Structural Principles in Robo Activation and Auto-Inhibition

This is the author accepted manuscript. The final version is available from Elsevier (Cell Press) via the DOI in this record.Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos’ switch from “off” to “on” states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1–8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.ICRFIS

Multimodal single-molecule microscopy with continuously controlled spectral resolution

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. Building on spectral imaging schemes that encode color into a fixed spatial intensity distribution, here, we introduce continuously controlled spectral-resolution (CoCoS) microscopy, which allows the spectral resolution of the system to be adjusted in real-time. By optimizing the spectral resolution for each experiment, we achieve maximal sensitivity and throughput, allowing for single-frame acquisition of multiple color channels with single-molecule sensitivity and 140-fold larger fields of view compared with previous super-resolution spectral imaging techniques. Here, we demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer, and multicolor single-particle tracking in live neurons, using a range of samples and 12 distinct fluorescent markers. A simple add-on allows CoCoS to be integrated into existing fluorescence microscopes, rendering spectral imaging accessible to the wider scientific community

CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling

Regulation of axon guidance and pruning of inappropriate synapses by class 3 semaphorins are key to the development of neural circuits. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating semaphorin 3A (Sema3A) signaling;however, nothing is known about its role in synapse pruning. Here, using newly generated crmp2(-/-) mice we demonstrate that CRMP2 has a moderate effect on Sema3A-dependent axon guidance in vivo, and its deficiency leads to a mild defect in axon guidance in peripheral nerves and the corpus callosum. Surprisingly, crmp2(-/-) mice display prominent defects in stereotyped axon pruning in hippocampus and visual cortex and altered dendritic spine remodeling, which is consistent with impaired Sema3F signaling and with models of autism spectrum disorder (ASD). We demonstrate that CRMP2 mediates Sema3F signaling in primary neurons and that crmp2(-/-) mice display ASD-related social behavior changes in the early postnatal period as well as in adults. Together, we demonstrate that CRMP2 mediates Sema3F-dependent synapse pruning and its dysfunction shares histological and behavioral features of ASD

Positional Information Generated by Spatially Distributed Signaling Cascades

The temporal and stationary behavior of protein modification cascades has been extensively studied, yet little is known about the spatial aspects of signal propagation. We have previously shown that the spatial separation of opposing enzymes, such as a kinase and a phosphatase, creates signaling activity gradients. Here we show under what conditions signals stall in the space or robustly propagate through spatially distributed signaling cascades. Robust signal propagation results in activity gradients with long plateaus, which abruptly decay at successive spatial locations. We derive an approximate analytical solution that relates the maximal amplitude and propagation length of each activation profile with the cascade level, protein diffusivity, and the ratio of the opposing enzyme activities. The control of the spatial signal propagation appears to be very different from the control of transient temporal responses for spatially homogenous cascades. For spatially distributed cascades where activating and deactivating enzymes operate far from saturation, the ratio of the opposing enzyme activities is shown to be a key parameter controlling signal propagation. The signaling gradients characteristic for robust signal propagation exemplify a pattern formation mechanism that generates precise spatial guidance for multiple cellular processes and conveys information about the cell size to the nucleus