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.

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.

Sleep Disorders in Patients With Posttraumatic Stress Disorder.

A growing body of evidence supports a bidirectional relationship between posttraumatic stress disorder (PTSD) and sleep disturbances. Fragmented sleep induced by sleep-related breathing disorders, insomnia, and nightmares impacts recovery and treatment outcomes and worsens PTSD symptoms. Despite recent attention, management of these disorders has been unrewarding in the setting of PTSD. This review summarizes the evidence for empirically supported treatments of these sleep ailments, including psychotherapeutic and pharmacologic interventions, as it relates to PTSD. Recent advances in positive airway pressure technology have made treatment of OSA more acceptable; however, adherence to CPAP therapy presents a substantial challenge. Concomitant insomnia, which engenders psychiatric and medical conditions, including depression, suicide, and alcohol and substance abuse, can be managed with cognitive behavioral therapy. Hypnotic agents are considered an alternative therapy, but concerns about adverse events and lack of high-level evidence supporting their efficacy in PTSD treatment have limited their use to resistant cases or as adjuncts to behavioral therapy when the response is less than desirable. Intrusion of nightmares can complicate PTSD treatment and exert serious strain on social, occupational, and marital relations. Imagery rehearsal therapy has shown significant reduction in nightmare intensity and frequency. The success of noradrenergic blocking agents has not been consistent among studies, with one-half reporting treatment failure. An integrated stepped care approach that includes components of both behavioral and pharmacologic interventions customized to patients' sleep-maladaptive behaviors may offer a solution to delivering accessible, effective, and efficient services for individuals with PTSD.