eLife最新文献

The major facilitator superfamily (MFS) transporters play significant roles in human health and disease. Salmonella enterica serovar Typhimurium melibiose permease (MelB_St) catalyzes the symport of galactosides with Na⁺, H⁺, or Li⁺ and is a prototype of MFS transporters. We published the structures of MelB_St in both inward- and outward-facing conformations, bound to galactoside or Na⁺, and proposed that positive cooperativity of the co-transported solutes is crucial for the symport mechanism. Here, we elucidated the underlying mechanisms by analyzing MelB_St dynamics and the effects of melibiose, Na⁺, or both using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We also refined the determinants of sugar recognition by solving the crystal structures of a uniporter D59C MelB_St complexed with melibiose and other sugars, and by identifying a critical water molecule involved in sugar recognition. Our integrated studies, combining structures, HDX-MS, and molecular dynamics simulations, support the conclusion that sugar-binding affinity is directly correlated with protein dynamics. Na⁺ acts as an allosteric activator, reducing the flexibility of dynamic residues in the sugar-binding site and in the cytoplasmic gating salt-bridge network, thereby increasing sugar-binding affinity. This study provides a molecular-level framework of the symport mechanism that could serve as a general model for cation-coupled symporters.

Legionella pneumophila is an opportunistic bacterial pathogen that causes Legionnaires' disease. To establish an intracellular niche conducive to replication, L. pneumophila translocates a diverse array of effector proteins that manipulate various host cellular processes, including the actin cytoskeleton. In a screen for effectors that alter actin dynamics, we identified a Legionella effector, Lfat1 (lpg1387), which colocalizes with the actin cytoskeleton in eukaryotic cells. Lfat1 specifically binds F-actin through a novel actin-binding domain (ABD). High-resolution cryo-electron microscopy (Cryo-EM) analysis revealed that this ABD forms a long α-helix hairpin, with its tip interacting with subdomains I and II of two adjacent actin molecules within the F-actin filament. Interestingly, while individual α-helices of the hairpin fail to bind F-actin, co-expression as separate fusion proteins restores binding activity. Furthermore, we demonstrated that Lfat1 exhibits lysine fatty acyltransferase (KFAT) activity, targeting host small GTPases. These findings establish a foundation for studying the KFAT family of bacterial toxins and uncover a novel F-actin-binding motif, providing an alternative F-actin marker with notable flexibility.

Avian influenza (AI) is a highly contagious viral disease affecting poultry and wild water birds, posing significant global challenges due to its high mortality rates and economic impacts. Highly pathogenic avian influenza (HPAI) outbreaks, particularly those caused by H5N1 and its variants, have surged since 1959. The HPAI H5N1 clade 2.3.4.4b viruses have notably expanded their geographical reach, affecting numerous countries, diverse avian species, and now mammals. Using an ecological niche modelling approach, this study aims to elucidate the environmental factors associated with increased HPAI H5 cases since 2020, investigate potential shifts in ecological niches, and predict new areas suitable for viral circulation. We developed ecological niche models for HPAI cases in wild and domestic birds across two distinct periods: 2015-2020 and 2020-2022. Key environmental predictors include chicken and duck population density, human density, distance to water bodies, and land cover variables. Post-2020, we observe increased relative influence of predictors such as intensive chicken population density and cultivated vegetation. Risk maps reveal notable ecological suitability for HPAI H5 circulation in Europe, Asia, and the Americas, with significant expansions of at-risk areas post-2020. Wild bird H5 occurrences appear primarily correlated with urban areas and open water regions. Our analyses also highlight a potential shift in affected wild bird species diversity, with more avian species, particularly sea birds, impacted post-2020. Overall, these results further contribute to the understanding of HPAI epidemiology and identify regions where surveillance and control measures should be prioritised.

Limbs execute diverse actions coordinated by the nervous system through multiple motor programs. The basic architecture of motor neurons that activate muscles which articulate joints for antagonistic flexion and extension movements is conserved from flies to vertebrates. While excitatory premotor circuits are expected to establish sets of leg motor neurons that work together, our study uncovered an instructive role for inhibitory circuits - including their ability to generate rhythmic leg movements. Using electron microscopy data in the Drosophila nerve cord, we categorized ~120 GABAergic inhibitory neurons from the 13 A and 13B hemilineages into classes based on similarities in morphology and connectivity. By mapping their connections, we uncovered pathways for inhibiting specific groups of motor neurons, disinhibiting antagonistic counterparts, and inducing alternation between flexion and extension. We tested the function of specific inhibitory neurons through optogenetic activation and silencing, using high-resolution quantitative analysis of leg movements during grooming. We combined findings from anatomical and behavioral analyses to construct a computational model that can reproduce major aspects of the observed behavior, demonstrating that these premotor inhibitory circuits can generate rhythmic leg movements.

Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry in vivo, or for optimally informative disease modeling and/or therapeutic screening in vitro, it is important to reproduce the type or subtype of neurons involved. No such appropriate in vitro models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor Sox6 is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that Sox6 suppresses a latent neurogenic program by repressing proneural Neurog2 expression by progenitors. We FACS-purify these progenitors from postnatal mouse cortex and establish a culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating Neurog2, Fezf2, while antagonizing Olig2 with VP16:Olig2). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons in vivo. They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed Neurog2-driven differentiation. Neurons induced by Neurog2 instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from cortical progenitors sets a precedent and foundation for in vitro mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.

Fever raises body temperature (T_b) from ~37°C to beyond 38.4°C to combat pathogens. While generally well tolerated below 40°C, in rare cases, fever can abnormally elevate neural activity and induce seizures in neurotypical children aged 2-5 years. This study investigates the mechanisms by which neuronal activity is maintained and stabilized during exposure to fever-range temperatures. Recordings of layer (L)4-evoked spiking in L2/3 pyramidal neurons (PNs) of mouse somatosensory cortex revealed four outcomes as temperature increased from 30°C to 36°C and 39°C (fever-range): neurons remained inactive, stayed active, ceased activity, or initiated activity. Roughly equal proportions of neurons ceased or initiated spiking, making the subset of 'STAY' PNs, those that remain active across temperatures, crucial for maintaining stable cortical output. STAY PNs were more prevalent at younger postnatal ages. Their firing stability was supported by a distinct ion channel composition, including the thermosensitive channel TRPV3, which enables continued spiking by adjusting depolarization to meet spike threshold. Intracellular blockade of TRPV3, but not TRPV4, significantly reduced the proportion of STAY PNs and suppressed spiking at 39°C. Moreover, in Trpv3^-/- mice, temperature increases to 39°C reduced both spiking and post-synaptic potential amplitude, and these mice exhibited a delayed seizure onset. Together, these findings suggest that TRPV3 contributes to the preservation of cortical activity during fever.

Striatal spiny-projection neurons (SPNs) integrate glutamatergic inputs from the motor cortex and thalamus with neuromodulatory signals to regulate motor output. In vivo Ca²⁺ imaging has demonstrated that spatially overlapping ensembles of direct and indirect pathway SPNs (dSPNs, iSPNs) are co-active during spontaneous movement. This co-activity is statistically greater among nearby neurons, correlates with behavioral state, and changes in an SPN-type-specific manner under pathological conditions. How this co-activity arises and whether it is important for movement are not well understood. Co-activity likely arises from shared excitatory inputs, the strength of which are scaled by well-characterized mechanisms of synaptic plasticity. Here, we show that the Group I metabotropic glutamate receptor 5 (mGluR5), which regulates the strength of corticostriatal synapses, is a key mediator of behaviorally relevant SPN co-activity. Pharmacological modulation of mGluR5 signaling bidirectionally altered movement and co-activity, but not the absolute level of activity in dSPNs. Targeted deletion of mGluR5 in dSPNs recapitulated the effects on spatiotemporal neural dynamics and movement, consistent with a striatum-specific effect of mGluR5 modulation. Targeted deletion of mGluR5 also produced changes in the synaptic properties of dSPNs. Separate from any effects on overall activity, our results show that excitatory synaptic modulation influences motor function by coordinating the spatial co-activation of dSPNs in vivo.

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed 'microglia replacement'. To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Neuronal aging and neurodegenerative diseases are accompanied by proteostasis collapse, while the cellular factors that trigger it have not been identified. Impaired mitochondrial transport in the axon is another feature of aging and neurodegenerative diseases. Using Drosophila, we found that genetic depletion of axonal mitochondria causes dysregulation of protein degradation. Axons with mitochondrial depletion showed abnormal protein accumulation and autophagic defects. Lowering neuronal ATP levels by blocking glycolysis did not reduce autophagy, suggesting that autophagic defects are associated with mitochondrial distribution. We found that eIF2β was increased by the depletion of axonal mitochondria via proteome analysis. Phosphorylation of eIF2α, another subunit of eIF2, was lowered, and global translation was suppressed. Neuronal overexpression of eIF2β phenocopied the autophagic defects and neuronal dysfunctions, and lowering eIF2β expression rescued those perturbations caused by depletion of axonal mitochondria. These results indicate the mitochondria-eIF2β axis maintains proteostasis in the axon, of which disruption may underlie the onset and progression of age-related neurodegenerative diseases.

Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a molecule that promotes adaptive ER proteostasis remodeling through mechanisms including preferential activation of the ATF6 signaling arm of the unfolded protein response (Plate et al., 2016). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a potential molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated α1-antitrypsin (A1AT) variant and an epilepsy-associated GABA_A receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically diverse protein misfolding disorders.