Context-specific stress causes compartmentalized SARM1 activation and local degeneration in cortical neurons.

SARM1 is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semi-automated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed sex mouse primary cortical neurons and male human iPSC derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection and SARM1 activation is limited to the axonal compartment distal of the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor.Significance Statement SARM1 is an important regulator of active axon degeneration after injury in the peripheral nervous system. Here we show that SARM1 can also be activated by a number of different cellular stressors in cortical neurons of the central nervous system. Loss or activation of SARM1 does not cause large scale changes in global protein homeostasis. However, context-dependent SARM1 activation is localized to specific neuronal compartments and results in localized degeneration of axons. Understanding which cell stress pathways are responsible for driving degeneration of distinct neuronal compartments under what cellular stress conditions and in which neuronal subtypes, will inform development of neurodegenerative disease therapeutics.

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity.

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

TMEM106B reduction does not rescue GRN deficiency in iPSC-derived human microglia and mouse models.

Heterozygous mutations in the granulin (GRN) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn -/-mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn -/-mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD-GRN.

Polarized microtubule remodeling transforms the morphology of reactive microglia and drives cytokine release.

Microglial reactivity is a pathological hallmark in many neurodegenerative diseases. During stimulation, microglia undergo complex morphological changes, including loss of their characteristic ramified morphology, which is routinely used to detect and quantify inflammation in the brain. However, the underlying molecular mechanisms and the relation between microglial morphology and their pathophysiological function are unknown. Here, proteomic profiling of lipopolysaccharide (LPS)-reactive microglia identifies microtubule remodeling pathways as an early factor that drives the morphological change and subsequently controls cytokine responses. We find that LPS-reactive microglia reorganize their microtubules to form a stable and centrosomally-anchored array to facilitate efficient cytokine trafficking and release. We identify cyclin-dependent kinase 1 (Cdk-1) as a critical upstream regulator of microtubule remodeling and morphological change in-vitro and in-situ. Cdk-1 inhibition also rescues tau and amyloid fibril-induced morphology changes. These results demonstrate a critical role for microtubule dynamics and reorganization in microglial reactivity and modulating cytokine-mediated inflammatory responses.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-ray Scattering.

With three FDA-approved products, lipid nanoparticles (LNPs) are under intensive development for delivering wide-ranging nucleic acid therapeutics. A significant challenge for LNP development is insufficient understanding of structure-activity relationship (SAR). Small changes in chemical composition and process parameters can affect LNP structure, significantly impacting performance in vitro and in vivo. The choice of polyethylene glycol lipid (PEG-lipid), one of the essential lipids for LNP, has been proven to govern particle size. Here we find that PEG-lipids can further modify the core organization of antisense oligonucleotide (ASO)-loaded LNPs to govern its gene silencing activity. Furthermore, we also have found that the extent of compartmentalization, measured by the ratio of disordered vs ordered inverted hexagonal phases within an ASO-lipid core, is predictive of in vitro gene silencing. In this work, we propose that a lower ratio of disordered/ordered core phases correlates with stronger gene knockdown efficacy. To establish these findings, we developed a seamless high-throughput screening approach that integrated an automated LNP formulation system with structural analysis by small-angle X-ray scattering (SAXS) and in vitro TMEM106b mRNA knockdown assessment. We applied this approach to screen 54 ASO-LNP formulations while varying the type and concentration of PEG-lipids. Representative formulations with diverse SAXS profiles were further visualized using cryogenic electron microscopy (cryo-EM) to help structural elucidation. The proposed SAR was built by combining this structural analysis with in vitro data. Our integrated methods, analysis, and resulting findings on PEG-lipid can be applied to rapidly optimize other LNP formulations in a complex design space.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection.

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) are required for microtubule polarity in Caenorhabditis elegans dendrites.

The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.

Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing.

Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. As one of the constituent lipids on LNP surfaces, PEGylated lipids (PEG-lipids) play an important role in defining LNP physicochemical properties and biological interactions. Previous studies indicate that LNP performance is modulated by tuning PEG-lipid parameters including PEG size and architecture, carbon tail type and length, as well as the PEG-lipid molar ratio in LNPs. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables. To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). A library of 54 ASO-LNP formulations with distinct PEG-lipid compositions was prepared using a liquid handling robot and assessed for their physiochemical properties as well as gene silencing efficacy in murine cortical neurons. Our results show that the molar ratio of anionic PEG-lipid in LNPs regulates particle size and PEG-lipid carbon tail length controls ASO-LNP gene silencing activity. ASO-LNPs formulated using PEG-lipids with optimal carbon tail lengths achieved up to 5-fold lower mRNA expression in neurons as compared to naked ASO. Representative ASO-LNP formulations were further characterized using dose-response curves and small-angle X-ray scattering to understand structure-activity relationships. Identified hits were also tested for efficacy in primary murine microglia and were scaled-up using a microfluidic formulation technique, demonstrating a smooth translation of ASO-LNP properties and in vitro efficacy. The reported HTS workflow can be used to screen additional multivariate parameters of LNPs with significant time and material savings, therefore guiding the selection and scale-up of optimal formulations for nucleic acid delivery to a variety of cellular targets.

Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration.

Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.

Organization and dynamics of the cortical complexes controlling insulin secretion in ß-cells.

Insulin secretion in pancreatic ß-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5ß (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5ß or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Inducible manipulation of motor-cargo interaction using engineered kinesin motors.

Molecular motors drive long-range intracellular transport of various vesicles and other cargoes within a cell. Identifying which kinesin motors interact with which type of transport vesicles has been challenging, especially in complex neuronal cells. Here, we present a highly adaptable toolbox of engineered kinesin motors to control and interrogate the selectivity and regulation of cargo transport with acute chemical induction. Selectivity of cargo-motor interaction can be addressed by systematic screening of a library of kinesin tails and neuronal cargoes. Additionally, our toolbox can be used to study kinesin-cargo regulatory mechanisms, and we found that cargo trafficking by KIF16B is regulated by its PX domain. Furthermore, our toolbox enables acute manipulation of polarized trafficking in living neurons by steering transport into axons or dendrites. Engineering kinesin motors provides a powerful tool to map the specificity of interactions between kinesin and cargoes, manipulate polarized transport and investigate cargo-motor interaction modes.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

Specific KIF1A-adaptor interactions control selective cargo recognition.

Intracellular transport in neurons is driven by molecular motors that carry many different cargos along cytoskeletal tracks in axons and dendrites. Identifying how motors interact with specific types of transport vesicles has been challenging. Here, we use engineered motors and cargo adaptors to systematically investigate the selectivity and regulation of kinesin-3 family member KIF1A-driven transport of dense core vesicles (DCVs), lysosomes, and synaptic vesicles (SVs). We dissect the role of KIF1A domains in motor activity and show that CC1 regulates autoinhibition, CC2 regulates motor dimerization, and CC3 and PH mediate cargo binding. Furthermore, we identify that phosphorylation of KIF1A is critical for binding to vesicles. Cargo specificity is achieved by specific KIF1A adaptors; MADD/Rab3GEP links KIF1A to SVs, and Arf-like GTPase Arl8A mediates interactions with DCVs and lysosomes. We propose a model where motor dimerization, posttranslational modifications, and specific adaptors regulate selective KIF1A cargo trafficking.

ER - lysosome contacts at a pre-axonal region regulate axonal lysosome availability.

Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the endoplasmic reticulum (ER) and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule - lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule - lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function.

Multiple layers of spatial regulation coordinate axonal cargo transport.

Nerve axons are shaped similar to long electric wires to quickly transmit information from one end of the body to the other. To remain healthy and functional, axons depend on a wide range of cellular cargos to be transported from the neuronal cell body to its distal processes. Because of the extended distance, a sophisticated and well-organized trafficking network is required to move cargos up and down the axon. Besides motor proteins driving cargo transport, recent data revealed that subcellular membrane specializations, including the axon initial segment at the beginning of the axon and the membrane-associated periodic skeleton, which extends throughout the axonal length, are important spatial regulators of cargo traffic. In addition, tubulin modifications and microtubule-associated proteins present along the axonal cytoskeleton have been proposed to bias cargo movements. Here, we discuss the recent advances in understanding these multiple layers of regulatory mechanisms controlling axonal transport.

Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

Neurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

The mechanism of kinesin inhibition by kinesin-binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain's tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.