Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.

The neuropeptidergic connectome of C. elegans.

Efforts are ongoing to map synaptic wiring diagrams, or connectomes, to understand the neural basis of brain function. However, chemical synapses represent only one type of functionally important neuronal connection; in particular, extrasynaptic, "wireless" signaling by neuropeptides is widespread and plays essential roles in all nervous systems. By integrating single-cell anatomical and gene-expression datasets with biochemical analysis of receptor-ligand interactions, we have generated a draft connectome of neuropeptide signaling in the C. elegans nervous system. This network is characterized by high connection density, extended signaling cascades, autocrine foci, and a decentralized topology, with a large, highly interconnected core containing three constituent communities sharing similar patterns of input connectivity. Intriguingly, several key network hubs are little-studied neurons that appear specialized for peptidergic neuromodulation. We anticipate that the C. elegans neuropeptidergic connectome will serve as a prototype to understand how networks of neuromodulatory signaling are organized.

Functional Organization of Glycolytic Metabolon on Mitochondria.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo.

Programmed Cell Death Modifies Neural Circuits and Tunes Intrinsic Behavior.

Programmed cell death is a common feature of animal development. During development of the C. elegans hermaphrodite, programmed cell death (PCD) removes 131 cells from stereotyped positions in the cell lineage, mostly in neuronal lineages. Blocking cell death results in supernumerary "undead" neurons. We find that undead neurons can be wired into circuits, can display activity, and can modify specific behaviors. The two undead RIM-like neurons participate in the RIM-containing circuit that computes movement. The addition of these two extra neurons results in animals that initiate fewer reversals and lengthens the duration of those reversals that do occur. We describe additional behavioral alterations of cell-death mutants, including in turning angle and pharyngeal pumping. These findings reveal that, like too much PCD, too little PCD can modify nervous system function and animal behavior.

A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans.

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport.

Neurexin and frizzled intercept axonal transport at microtubule minus ends to control synapse formation.

Synapse formation is locally determined by transmembrane proteins, yet synaptic material is synthesized remotely and undergoes processive transport in axons. How local synaptogenic signals intercept synaptic cargo in transport to promote its delivery and synapse formation is unknown. We found that the control of synaptic cargo delivery at microtubule (MT) minus ends mediates pro- and anti-synaptogenic activities of presynaptic neurexin and frizzled in C. elegans and identified the atypical kinesin VAB-8/KIF26 as a key molecule in this process. VAB-8/KIF26 levels at synaptic MT minus ends are controlled by frizzled and neurexin; loss of VAB-8 mimics neurexin mutants or frizzled hyperactivation, and its overexpression can rescue synapse loss in these backgrounds. VAB-8/KIF26 is required for the synaptic localization of other minus-end proteins and promotes the pausing of retrograde transport to allow delivery to synapses. Consistently, reducing retrograde transport rescues synapse loss in vab-8 and neurexin mutants. These results uncover a mechanistic link between synaptogenic signaling and axonal transport.

Activation of the CaMKII-Sarm1-ASK1-p38 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria.

Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In Caenorhabditis elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 activates the conserved Sarm1/TIR-1-ASK1/NSY-1-p38 MAPK pathway and eventually the transcription factor CEBP-1 to protect against degeneration. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated, and uncover a surprising neuroprotective role for the Sarm1-p38 MAPK pathway in this context.

Within the cell are various compartments that carry out specific roles. This includes the mitochondria, which are responsible for generating the chemical energy that powers the cell. Some of the most power-hungry cells are nerve cells, which have long, slender projections called axons that relay signals from one part of the nervous system to another. If the mitochondria do not work properly, the axons break down which can lead to neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Previous studies showed that defects in how mitochondria are transported cause axons in the roundworm Caenorhabditis elegans to spontaneously deteriorate with age. These transparent worms are often used to study biological questions related to the nervous system. Here, Ding et al. have used this model organism to identify a molecular pathway that stops axons from degenerating. Random mutations were introduced into the genome of C. elegans that are unable to transport mitochondria in to their axons. Ding et al. then searched for mutant strains that still had intact axons despite this mitochondrial defect. This revealed that mutations that activate a protein called CaMKII stop axons from breaking down. Further experiments showed that CAMKII does this by switching on a series of signals, including the protein Sarm1, that eventually turn on another protein that suppresses degeneration. The protective role of Sarm1 is surprising given that this protein has been shown to promote axon degeneration after injury in flies and mammals. This suggests that the gene for Sarm1 as well as others likely play different roles depending on the context in which they are activated. These findings could help researchers identify new drug targets and strategies for treating neurodegenerative diseases caused by mitochondrial defects. However, further work is needed to see if this newly discovered pathway works the same way in other model systems, such as flies and mammals.

rab-27 acts in an intestinal pathway to inhibit axon regeneration in C. elegans.

Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.

Molecular topography of an entire nervous system.

We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of ∼23 neuropeptide genes and ∼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.

In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system.

The generation of the enormous diversity of neuronal cell types in a differentiating nervous system entails the activation of neuron type-specific gene batteries. To examine the regulatory logic that controls the expression of neuron type-specific gene batteries, we interrogate single cell expression profiles of all 118 neuron classes of the Caenorhabditis elegans nervous system for the presence of DNA binding motifs of 136 neuronally expressed C. elegans transcription factors. Using a phylogenetic footprinting pipeline, we identify cis-regulatory motif enrichments among neuron class-specific gene batteries and we identify cognate transcription factors for 117 of the 118 neuron classes. In addition to predicting novel regulators of neuronal identities, our nervous system-wide analysis at single cell resolution supports the hypothesis that many transcription factors directly co-regulate the cohort of effector genes that define a neuron type, thereby corroborating the concept of so-called terminal selectors of neuronal identity. Our analysis provides a blueprint for how individual components of an entire nervous system are genetically specified.

A head-to-head comparison of ribodepletion and polyA selection approaches for Caenorhabditis elegans low input RNA-sequencing libraries.

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in Caenorhabditis elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Furthermore, some common resources, such as sequence-specific kits for removing ribosomal RNA, are not optimized for nonmammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

A Functional Non-coding RNA Is Produced from xbp-1 mRNA.

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.

The stress-responsive gene GDPGP1/mcp-1 regulates neuronal glycogen metabolism and survival.

Maladaptive responses to stress might play a role in the sensitivity of neurons to stress. To identify novel cellular responses to stress, we performed transcriptional analysis in acutely stressed mouse neurons, followed by functional characterization in Caenorhabditis elegans. In both contexts, we found that the gene GDPGP1/mcp-1 is down-regulated by a variety of stresses. Functionally, the enzyme GDPGP1/mcp-1 protects against stress. Knockdown of GDPGP1 in mouse neurons leads to widespread neuronal cell death. Loss of mcp-1, the single homologue of GDPGP1 in C. elegans, leads to increased degeneration of GABA neurons as well as reduced survival of animals following environmental stress. Overexpression of mcp-1 in neurons enhances survival under hypoxia and protects against neurodegeneration in a tauopathy model. GDPGP1/mcp-1 regulates neuronal glycogen levels, indicating a key role for this metabolite in neuronal stress resistance. Together, our data indicate that down-regulation of GDPGP1/mcp-1 and consequent loss of neuronal glycogen is a maladaptive response that limits neuronal stress resistance and reduces survival.

Mechanisms of injury-induced axon degeneration.

Injury-induced axon degeneration in model organisms and cell culture has emerged as an area of growing interest due to its experimental tractability and to the promise of identifying conserved mechanisms that mediate axon loss in human disease. Injury-induced axon degeneration is also observed within the well-studied process of Wallerian degeneration, a complex phenomenon triggered by axon injury to peripheral nerves in mammals. Recent studies have led to the identification of key molecular components of injury-induced axon degeneration. Axon survival factors, such as NMNAT2, act to protect injured axons from degeneration. By contrast, factors such as SARM1, MAPK, and PHR1 act to promote degeneration. The coordinated activity of these factors determines axon fate after injury. Since axon loss is an early feature of neurodegenerative diseases, it is possible that understanding the molecular mechanism of injury-induced degeneration will lead to new treatments for axon loss in neurodegenerative disease. Here, we discuss the critical pathways for injury-induced axon degeneration across species with an emphasis on their interactions in an integrated signaling network.

Aberrant information transfer interferes with functional axon regeneration.

Functional axon regeneration requires regenerating neurons to restore appropriate synaptic connectivity and circuit function. To model this process, we developed an assay in Caenorhabditis elegans that links axon and synapse regeneration of a single neuron to recovery of behavior. After axon injury and regeneration of the DA9 neuron, synapses reform at their pre-injury location. However, these regenerated synapses often lack key molecular components. Further, synaptic vesicles accumulate in the dendrite in response to axon injury. Dendritic vesicle release results in information misrouting that suppresses behavioral recovery. Dendritic synapse formation depends on dynein and jnk-1. But even when information transfer is corrected, axonal synapses fail to adequately transmit information. Our study reveals unexpected plasticity during functional regeneration. Regeneration of the axon is not sufficient for the reformation of correct neuronal circuits after injury. Rather, synapse reformation and function are also key variables, and manipulation of circuit reformation improves behavioral recovery.

γ-Neurexin and Frizzled Mediate Parallel Synapse Assembly Pathways Antagonized by Receptor Endocytosis.

Synapse formation defines neuronal connectivity and is thus essential for neuronal circuit assembly. Trans-synaptic interactions of cell adhesion molecules are thought to induce synapse assembly. Here we demonstrate that a recently discovered and conserved short form of neurexin, γ-neurexin, which lacks canonical extracellular domains, is nonetheless sufficient to promote presynaptic assembly in the nematode C. elegans. γ- but not α-neurexin is required for assembling active zone components, recruiting synaptic vesicles, and clustering calcium channels at release sites to promote evoked synaptic transmission. Furthermore, we find that neurexin functions in parallel with the transmembrane receptor Frizzled, as the absence of both proteins leads to an enhanced phenotype-the loss of most synapses. Frizzled's pro-synaptogenic function is independent of its ligand, Wnt. Wnt binding instead eliminates synapses by inducing Frizzled's endocytosis and the downregulation of neurexin. These results reveal how pro- and anti-synaptogenic factors converge to precisely sculpt circuit formation in vivo.