First Detection of Algal Caribbean Ciguatoxin in Amberjack Causing Ciguatera Poisoning in the Canary Islands (Spain).

Ciguatera Poisoning (CP) is an illness associated with the consumption of fish contaminated with potent natural toxins found in the marine environment, commonly known as ciguatoxins (CTXs). The risk characterization of CP has become a worldwide concern due to the widespread expansion of these natural toxins. The identification of CTXs is hindered by the lack of commercially available reference materials. This limitation impedes progress in developing analytical tools and conducting toxicological studies essential for establishing regulatory levels for control. This study focuses on characterizing the CTX profile of an amberjack responsible for a recent CP case in the Canary Islands (Spain), located on the east Atlantic coast. The exceptional sensitivity offered by Capillary Liquid Chromatography coupled with High-Resolution Mass Spectrometry (cLC-HRMS) enabled the detection, for the first time in fish contaminated in the Canary Islands, of traces of an algal ciguatoxin recently identified in G. silvae and G. caribeaus from the Caribbean Sea. This algal toxin was structurally characterized by cLC-HRMS being initially identified as C-CTX5. The total toxin concentration of CTXs was eight times higher than the guidance level proposed by the Food and Drug Administration (0.1 ng C-CTX1/g fish tissue), with C-CTX1 and 17-hydroxy-C-CTX1 as major CTXs.

Neutrophil-inflicted vasculature damage suppresses immune-mediated optic nerve regeneration.

In adult mammals, injured retinal ganglion cells (RGCs) fail to spontaneously regrow severed axons, resulting in permanent visual deficits. Robust axon growth, however, is observed after intra-ocular injection of particulate ß-glucan isolated from yeast. Blood-borne myeloid cells rapidly respond to ß-glucan, releasing numerous pro-regenerative factors. Unfortunately, the pro-regenerative effects are undermined by retinal damage inflicted by an overactive immune system. Here, we demonstrate that protection of the inflamed vasculature promotes immune-mediated RGC regeneration. In the absence of microglia, leakiness of the blood-retina barrier increases, pro-inflammatory neutrophils are elevated, and RGC regeneration is reduced. Functional ablation of the complement receptor 3 (CD11b/integrin-αM), but not the complement components C1q-/- or C3-/-, reduces ocular inflammation, protects the blood-retina barrier, and enhances RGC regeneration. Selective targeting of neutrophils with anti-Ly6G does not increase axogenic neutrophils but protects the blood-retina barrier and enhances RGC regeneration. Together, these findings reveal that protection of the inflamed vasculature promotes neuronal regeneration.

RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages.

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

Targeted suppression of mTORC2 reduces seizures across models of epilepsy.

Epilepsy is a neurological disorder that poses a major threat to public health. Hyperactivation of mTOR complex 1 (mTORC1) is believed to lead to abnormal network rhythmicity associated with epilepsy, and its inhibition is proposed to provide some therapeutic benefit. However, mTOR complex 2 (mTORC2) is also activated in the epileptic brain, and little is known about its role in seizures. Here we discover that genetic deletion of mTORC2 from forebrain neurons is protective against kainic acid-induced behavioral and EEG seizures. Furthermore, inhibition of mTORC2 with a specific antisense oligonucleotide robustly suppresses seizures in several pharmacological and genetic mouse models of epilepsy. Finally, we identify a target of mTORC2, Nav1.2, which has been implicated in epilepsy and neuronal excitability. Our findings, which are generalizable to several models of human seizures, raise the possibility that inhibition of mTORC2 may serve as a broader therapeutic strategy against epilepsy.

Reversible histone deacetylase activity catalyzes lysine acylation.

Starvation and low carbohydrate diets lead to the accumulation of the ketone body, ß-hydroxybutyrate (BHB), whose blood concentrations increase more than 10-fold into the millimolar range. In addition to providing a carbon source, BHB accumulation triggers lysine ß-hydroxybutyrylation (Kbhb) of proteins via unknown mechanisms. As with other lysine acylation events, Kbhb marks can be removed by histone deacetylases (HDACs). Here, we report that class I HDACs unexpectedly catalyze protein lysine modification with ß-hydroxybutyrate (BHB). Mutational analyses of the HDAC2 active site reveal a shared reliance on key amino acids for classical deacetylation and non-canonical HDAC-catalyzed ß-hydroxybutyrylation. Also consistent with reverse HDAC activity, Kbhb formation is driven by mass action and substrate availability. This reverse HDAC activity is not limited to BHB but also extends to multiple short-chain fatty acids. The reversible activity of class I HDACs described here represents a novel mechanism of PTM deposition relevant to metabolically-sensitive proteome modifications.

Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Adaptor protein AP-3 produces synaptic vesicles that release at high frequency by recruiting phospholipid flippase ATP8A1.

Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency.

The oncomodulin receptor ArmC10 enables axon regeneration in mice after nerve injury and neurite outgrowth in human iPSC-derived sensory neurons.

Oncomodulin (Ocm) is a myeloid cell-derived growth factor that enables axon regeneration in mice and rats after optic nerve injury or peripheral nerve injury, yet the mechanisms underlying its activity are unknown. Using proximity biotinylation, coimmunoprecipitation, surface plasmon resonance, and ectopic expression, we have identified armadillo-repeat protein C10 (ArmC10) as a high-affinity receptor for Ocm. ArmC10 deletion suppressed inflammation-induced axon regeneration in the injured optic nerves of mice. ArmC10 deletion also suppressed the ability of lesioned sensory neurons to regenerate peripheral axons rapidly after a second injury and to regenerate their central axons after spinal cord injury in mice (the conditioning lesion effect). Conversely, Ocm acted through ArmC10 to accelerate optic nerve and peripheral nerve regeneration and to enable spinal cord axon regeneration in these mouse nerve injury models. We showed that ArmC10 is highly expressed in human-induced pluripotent stem cell-derived sensory neurons and that exposure to Ocm altered gene expression and enhanced neurite outgrowth. ArmC10 was also expressed in human monocytes, and Ocm increased the expression of immune modulatory genes in these cells. These findings suggest that Ocm acting through its receptor ArmC10 may be a useful therapeutic target for nerve repair and immune modulation.

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles.

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

Non-specific recognition of histone modifications by H3K9bhb antibody.

Ketone bodies are short-chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative energy source for the brain, heart, and skeletal muscle. Beyond this metabolic role, ß-hydroxybutyrate (BHB), is gaining recognition as a signaling molecule. Lysine ß-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against ß-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s) that likely include acetylation. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Non-specific recognition of histone modifications by H3K9bhb antibody.

Ketone bodies are short chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative source of energy for the brain, heart, and skeletal muscle. Beyond this classical metabolic role, ß-hydroxybutyrate (BHB), is gaining recognition as a pleiotropic signaling molecule. Lysine ß-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This novel protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments, including the mitochondria and nucleus. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against the ß-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s), which are increased by deacetylation inhibition and include likely acetylations. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Characterization of the Ciguatoxin Profile in Fish Samples from the Eastern Atlantic Ocean using Capillary Liquid Chromatography-High Resolution Mass Spectrometry.

Ciguatera Poisoning is an emerging risk in the east Atlantic Ocean. Despite characterization efforts, the complete profile of ciguatoxin chemical species in these waters is still unknown. These efforts have been complicated by a lack of reference materials and scarcity of fish contaminated with high levels of ciguatoxins. Development and application of analytical methods with enhanced selectivity and sensitivity is essential for ciguatoxin characterization. Here, we developed an analytical characterization approach using capillary liquid chromatography coupled to high resolution mass spectrometry applied to reference materials obtained from ciguatoxin contaminated fish. Capillary LC coupled mass spectrometry resulted in increased sensitivity leading to the confirmation of C-CTX1 as the principal ciguatoxin present in these samples. We also detected and structurally characterized minor C-CTXs analogues consisting of C-CTX3/4, hydroxy-, didehydro-, and methoxy- metabolites.

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Capture, Release, and Identification of Newly Synthesized Proteins for Improved Profiling of Functional Translatomes.

New protein synthesis is regulated both at the level of mRNA transcription and translation. RNA-Seq is effective at measuring levels of mRNA expression, but techniques to monitor mRNA translation are much more limited. Previously, we reported results from O-propargyl-puromycin (OPP) labeling of proteins undergoing active translation in a 2-h time frame, followed by biotinylation using click chemistry, affinity purification, and on-bead digestion to identify nascent proteins by mass spectrometry (OPP-ID). As with any on-bead digestion protocol, the problem of nonspecific binders complicated the rigorous categorization of nascent proteins by OPP-ID. Here, we incorporate a chemically cleavable linker, Dde biotin-azide, into the protocol (OPP-IDCL) to provide specific release of modified proteins from the streptavidin beads. Following capture, the Dde moiety is readily cleaved with 2% hydrazine, releasing nascent polypeptides bearing OPP plus a residual C3H8N4 tag. When results are compared side by side with the original OPP-ID method, change to a cleavable linker led to a dramatic reduction in the number of background proteins detected in controls and a concomitant increase in the number of proteins that could be characterized as newly synthesized. We evaluated the method's ability to detect nascent proteins at various submilligram protein input levels and showed that, when starting with only 100 µg of protein, ∼1500 nascent proteins could be identified with low background. Upon treatment of K562 cells with MLN128, a potent inhibitor of the mammalian target of rapamycin, prior to OPP treatment, we identified 1915 nascent proteins, the majority of which were downregulated upon inhibitor treatment. Repressed proteins with log2 FC <-1 revealed a complex network of functionally interacting proteins, with the largest cluster associated with translational initiation. Overall, incorporation of the Dde biotin-azide cleavable linker into our protocol has increased the depth and accuracy of profiling of nascent protein networks.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production.

Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin ß1 Interactomes.

Importin ß1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin ß1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin ß1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

Comparative Proteomics of Coxiella like Endosymbionts (CLEs) in the Symbiotic Organs of Rhipicephalus sanguineus Ticks.

Maternally transmitted obligatory endosymbionts are found in the female gonads as well as in somatic tissue and are expected to provide missing metabolite to their hosts. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella-like endosymbionts (CLEs) of Rhipicephalus ticks. CLEs are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, but the role of CLEs in these tissues is unknown. To probe possible functions of CLEs, comparative proteomics was performed between Mt and Ov of R. sanguineus ticks. Altogether, a total of 580 and 614 CLE proteins were identified in Mt and Ov, respectively. Of these, 276 CLE proteins were more abundant in Mt, of which 12 were significantly differentially abundant. In Ov, 290 CLE proteins were more abundant, of which 16 were significantly differentially abundant. Gene Ontology analysis revealed that most of the proteins enriched in Mt are related to cellular metabolic functions and stress responses, whereas in Ov, the majority were related to cell proliferation suggesting CLEs function differentially and interdependently with host requirements specific to each organ. The results suggest Mt CLEs provide essential nutrients to its host and Ov CLEs promote proliferation and vertical transmission to tick progeny. IMPORTANCE Here we compare the Coxiella-like endosymbionts (CLEs) proteomes from Malpighian tubule (Mt) and the ovaries (Ov) of the brown dog tick Rhipicephalus sanguineus. Our results support the hypothesis that CLEs function interdependently with host requirements in each of the organs. The different functional specificity of CLE in the same host suggest that metabolic capabilities evolved according to the constrains imposed by the specific organ function and requirements. Our findings provide specific CLE protein targets that can be useful for future studies of CLE biology with a focus on tick population control.