Biology subjects

Mendes, A.

Publications and source records attributed to Mendes, A..

11 recordsLinked to original sources

bioRxiv2026

Pathogenic T-bet+ memory B cells worsen multiple sclerosis through myeloid cell activation

Depletion of B cells in multiple sclerosis (MS) is beneficial yet there is no defined pathogenic B cell subset in MS. Here, we demonstrate that T-bet+ memory B cells are rare in control human brain specimens but are found in MS lesions. Transfer of murine T-bet+ memory B cells into mice with ongoing experimental autoimmune encephalomyelitis (EAE), a model of MS, worsened their disability and demyelination. Microglia/macrophage density increased in central nervous system parenchyma even though B cells mostly remained in barriers. In culture, secreted factors from T-bet+ memory B cells promoted macrophage migration. IFN-{gamma} produced by T-bet+ memory B cells activated microglia to secrete chemokines that further enabled macrophage migration. Consistent with their age-associated elevation, conditional deletion of T-bet in B cells lowered EAE severity in aged but not young mice. We define T-bet+ memory B cells as pathogenic in EAE and MS through their IFN-{gamma}-facilitated interactions with microglia/macrophages.

immunology↗

bioRxiv2025

Non-synaptic exocytosis along the axon shaft and its regulation by the submembrane periodic skeleton

Neuronal communication relies on signaling molecules transferred via exo- and endocytosis throughout the brain. Historically, studies have focused on vesicle exo- and endocytosis and their release machinery at synapses, and much less is known about non-synaptic exocytosis. If and how vesicles can access the plasma membrane along the axonal shaft, overcoming the insulating layer of the membrane-associated periodic scaffold, remains unclear. Here, we used fast live-cell imaging of mature cultured hippocampal neurons expressing the vamp2-pHluorin reporter to map sponta-neous exocytosis along axons. We detected non-synaptic exocytic events along the axon shaft that concentrated at the axon initial segment. Perturbation of the membrane-associated actin-spectrin skeleton revealed its role in regulating shaft exocytosis, similarly to its recently demonstrated role in shaping axonal endocytosis. To visualize the nanoscale arrangement of exocytic locations, we developed a novel correlative live-cell/two - color, single-molecule localization microscopy (SMLM) approach. We observed that regions of exocytosis are devoid of the submembrane spectrin mesh, with these spectrin-free areas being spatially separated from the spectrin clearings that contain clathrin-coated pits. Overall, our work reveals a new process of spontaneous exocytosis along the axon shaft, and how the axonal submem-brane skeleton shapes a heterogeneous landscape that uniquely segregates vesicular trafficking events.

cell biology↗

bioRxiv2024

Induction of the ISR by AB5 subtilase cytotoxin drives IFN-I expression in pDCs via STING activation

We demonstrate that exposure to the AB5 subtilase cytotoxin (SubAB) induces the unfolded protein response (UPR) in human peripheral blood mononuclear cells, concomitant with a pro-inflammatory response across distinct cell subsets. Notably, SubAB selectively induces type-I interferon (IFN) expression in plasmacytoid dendritic cells, acting synergistically with Toll-like receptor 7 stimulation. The induction of type-I IFN in response to SubAB relies on stimulator of interferon genes (STING) activation, coupled with protein synthesis inhibition mediated by protein kinase R-like endoplasmic reticulum kinase and phosphorylation of the eukaryotic translation initiation factor 2 subunit-alpha. By impeding mRNA translation through the integrated stress response, SubAB precipitates the downregulation of the negative innate signaling feedback regulator Tax1-binding protein 1. This downregulation is necessary to unleash TANK-binding kinase 1 signaling associated with STING activation. These findings shed new light on how UPR-inducing conditions may regulate the immune system during infection or pathogenesis.

immunology↗

bioRxiv2024

Structural Repetition Detector: multi-scale quantitative mapping of molecular complexes through microscopy

From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReDs capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReDs utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.

cell biology↗

bioRxiv2024

VPS34-IN1 inhibits cap-mediated translation and synergizes with STING to drive type-I IFN expression in human plasmacytoid DCs

Inhibition of the phosphatidylinositol kinase vacuolar protein sorting 34 (VPS34) with the pharmacological compound VPS34-IN1 has a range of effects on the dynamics of endosomes. While VPS34 inhibition has been suggested as a potential therapeutic approach for treating certain cancers, our findings indicate that it has minimal cytotoxic effects on leukemic blastic plasmacytoid dendritic cell neoplasms (BPDCN). VPS34-IN1, however, interferes with plasmacytoid dendritic cells (pDCs) function by blocking the recruitment of serum and glucocorticoid-regulated kinase 3 (SGK3) to endosomes, which is shown to be necessary for Toll-like receptor 7 (TLR7) signaling. In a contrasting parallel, VPS34-IN1 triggers the activation of the stimulator of interferon genes (STING) and significantly enhances pDCs response to the STING agonist 23-cyclic guanosine monophosphate-adenosine monophosphate (23-cGAMP). This cooperative action with VPS34-IN1 leads to strongly increased expression of type-I interferons (IFNs), associated with an alteration of STING degradation and importantly, inhibition of cap-mediated mRNA translation. Inhibition of protein synthesis by VPS34-IN1 appears to be central to this synergy with STING activation, notably by compromising the expression of IFIT1/ISG56, a negative regulator of innate signaling. Thus, despite their limited toxicity towards different cancer lines, inhibitors targeting VPS34 and SGK3 may present promising compounds for controlling the expression of type-I IFNs in response to various microbial stimuli and pathological contexts. One-sentence summaryPharmacological inhibition of VPS34 affects multiple signaling pathways downstream of innate immunity receptors and consequently can inhibit or potentiate type-I Interferon induction according to the danger or microbial stimuli received by plasmacytoid DCs.

immunology↗

bioRxiv2022

Mannose-coupled AAV2: a second generation AAV vector for increased retinal gene therapy efficiency

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in immune reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

bioengineering↗

bioRxiv2022

Sensory neuron-associated macrophages proliferate in the sensory ganglia after peripheral nerve injury in a CX3CR1 signaling dependent manner

Resident macrophages are distributed across all tissues and are highly heterogeneous as a consequence of adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia which have been involved in different conditions, especially in neuropathic pain development and nerve regeneration. However, it is still under debate whether macrophages accumulation in the sensor ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or as a result of blood monocytes infiltration. Here, we confirmed that the number of macrophages increased in the DRGs after spared nerve injury (SNI) model in mice. By using different approaches, we found that the increase in the number of macrophages in the DRGs after SNI is mainly in consequence of the proliferation of resident CX3CR1+ macrophages but not due to infiltration of CCR2+ blood monocytes. These proliferating macrophages are the source of the production of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain and/or nerve regeneration.

immunology↗

bioRxiv2020

Developmentally regulated PERK activity renders dendritic cells insensitive to subtilase cytotoxin-induced integrated stress response

In stressed cells, phosphorylation of eukaryotic initiation factor 2 (eIF2) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response (ISR). We show here that dendritic cells (DCs) display unusually high eIF2 phosphorylation, which is mostly caused by a developmentally regulated activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2 levels, differentiated DCs display active protein synthesis and no signs of a chronic ISR. eIF2 phosphorylation does not majorly impact DC differentiation nor cytokines production. It is however important to adapt protein homeostasis to the variations imposed on DCs by the immune or physiological contexts. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by subtilase cytotoxin or upon DC stimulation with bacterial lipopolysaccharides. This is also exemplified by the influence of the actin cytoskeleton dynamics on eIF2 phosphorylation and the migratory deficit observed in PERK-deficient DCs.

immunology↗

bioRxiv2020

Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the {beta}-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

cancer biology↗

bioRxiv2019

A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

The discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

molecular biology↗

bioRxiv2018

Robustness of STDP to spike timing jitter

In Hebbian plasticity, neural circuits adjust their synaptic weights depending on patterned firing of action potential on either side of the synapse. Spike-timing-dependent plasticity (STDP) is an experimental implementation of Hebbs postulate that relies on the precise order and the millisecond timing of the paired activities in pre- and postsynaptic neurons. In recent years, STDP has attracted considerable attention in computational and experimental neurosciences. However, canonical STDP is assessed with deterministic (constant) spike timings and time intervals between successive pairings, thus exhibiting a regularity that strongly differs from the biological variability. Hence, the emergence of STDP from noisy neural activity patterns as expected in in vivo-like firing remains unresolved. Here, we used noisy STDP stimulations where the spike timing and/or the interval between successive pairings were jittered. We explored with a combination of experimental neurophysiology and mathematical modeling, the impact of jittering on three distinct forms of STDP at corticostriatal synapses: NMDAR-mediated tLTP, endocannabinoid-mediated tLTD and endocannabinoid-mediated tLTP. As the main result, we found a differential sensitivity to jittered spike timing: NMDAR-tLTP was highly fragile whereas endocannabinoid-plasticity (tLTD and tLTP) appeared more resistant. Moreover, when the frequency or the number of pairings was increased, NMDAR-tLTP became more robust and could be expressed despite strong jittering of the spike timing. Taken together, our results identify endocannabinoid-mediated plasticity as a robust form of STDP while the sensitivity to jitter of NMDAR-tLTP varies with activity frequency. This provides new insights into the mechanisms at play during the different phases of learning and memory and the emergence of Hebbian plasticity in in vivo-like firing.

neuroscience↗