Biology subjects

Villa, A.

Publications and source records attributed to Villa, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Do nanoplastics reshape microglial support of neuronal resilience? A study of microglial bioenergetics and microglia to neuron communication in vitro

Nanoplastics (NPs) are emerging environmental contaminants able to cross biological barriers, disrupt cellular and organelle homeostasis, and alter the brain microenvironment. This study investigated whether NPs affect microglia-neuron communication, a key mechanism underlying neuronal resilience, via the nuclear factor erythroid 2-like 2 (NFE2L2) pathway. Using an in vitro model, we evaluated the effects of polystyrene nanoplastics on microglial metabolic fitness and microglia-mediated neuronal stress responses. Increasing NP concentrations induced a dose-dependent biphasic effect. Low to intermediate concentrations increased intracellular adenosine triphosphate (ATP) levels in microglia and enhanced microglia-mediated activation of neuronal NFE2L2. In contrast, high NP concentration impaired microglial metabolism, reduced ATP availability, and decreased microglia-neuron communication. These findings indicate that NPs alter microglial energetic status and modulate neuroprotective signalling, potentially contributing to impaired neuron-microglia interactions and increased susceptibility to neurotoxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/732827v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@d33f79org.highwire.dtl.DTLVardef@ec4542org.highwire.dtl.DTLVardef@1b71ca3org.highwire.dtl.DTLVardef@b92ad4_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINanoplastics alter microglial metabolic fitness in vitro. C_LIO_LINanoplastics biphasically modulate microglial support of neurons. C_LIO_LIHigh nanoplastic concentration reduces microglial support to neurons. C_LIO_LIMicroglial bioenergetics may link nanoplastics to neuronal vulnerability. C_LI

pharmacology and toxicology↗

bioRxiv2026

Nmur1 and Cckar fail to support functional genetic access in adult dopamine neurons and challenge GPCR atlas assignments

Apuschkin et al. (2024) proposed a GPCR-based transcriptomic atlas for midbrain dopamine (DA) neuron subpopulations, including candidates such as Nmur1, Cckar, and Ffar4. To guide genetic targeting, these markers must reflect functional expression in adult DA neurons. Using in situ hybridization, Cre-dependent reporter lines, and both intracranial and systemic viral approaches, we find no evidence of adult Nmur1-mediated recombination in DA neurons, while Cckar-driven recombination is consistent with developmental expression only. Notably, Ffar4 expression overlaps extensively with Ntsr1 midbrain populations, indicating that it does not define a distinct DA neuron class. Furthermore, analysis of independent spatial transcriptomic datasets together with our MERFISH data shows that many proposed GPCR markers are not detectably expressed in adult DA neurons. These findings demonstrate that transcriptomic enrichment does not always yield reliable adult markers and highlight the need for functional validation prior to use in circuit targeting.

neuroscience↗

bioRxiv2026

Identification of ICAM-1-targeting DNA aptamers as a host-directed strategy to inhibit Human Rhinovirus infection

Exacerbations of respiratory viral infections significantly contribute to morbidity and healthcare burden. Among these viruses, Human Rhinoviruses (HRVs) are the most frequent causative agents of upper respiratory tract infections. To date, over 150 HRV serotypes have been identified, classified into three species: HRV-A, HRV-B, and HRV-C. No antiviral therapies are currently available against this viral family, largely due to the high serotype diversity and limited cross-protection. The major group of HRVs relies on the Intercellular Adhesion Molecule-1 (ICAM-1) receptor to infect airway epithelial cells, making ICAM-1 an attractive target for broad-spectrum therapeutic interventions. Here, we report the development of nucleic acid-based aptamers designed to disrupt ICAM-1-HRV binding and thereby prevent viral infection. Aptamers are single-stranded DNA molecules that fold into precise three-dimensional structures, enabling highly specific protein recognition. Using a Systematic Evolution of Ligands by EXponential Enrichment (SELEX) approach guided by a minimal peptide mimicking the ICAM-1 viral binding interface, a library of >1024 random single-stranded DNA sequences was screened. Through iterative rounds of selection, we identified eight candidate 77-nt DNA aptamers, which were subsequently evaluated for their potential using in silico and in vitro assays, as well as functional assays in human epithelial cells. From this strategy, two lead aptamers were selected that effectively inhibited HRV-A16 replication in a concentration-dependent manner, as measured by viral titers (TCID assay) and viral RNA quantification by RT-PCR. These findings demonstrate the potential of ICAM-1-targeting aptamers as antiviral agents capable of preventing HRV entry. By targeting a host receptor and creating a protective barrier at the cell surface, this approach may offer a broadly applicable strategy against multiple HRV serotypes, paving the way for the development of novel antiviral interventions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/717810v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@130d8org.highwire.dtl.DTLVardef@2dd09borg.highwire.dtl.DTLVardef@1da744eorg.highwire.dtl.DTLVardef@109ea5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Genetic Identification of Dopamine Neurons Required for Circadian Food Anticipatory Activity in Mice

Anticipating daily food availability is a conserved circadian behavior that persists even in animals lacking the suprachiasmatic nucleus, yet its neural substrates remain poorly defined. Previous studies implicated dopamine signaling in food anticipatory activity (FAA) but lacked the resolution to identify the responsible neuronal population. Here, we conditionally deleted tyrosine hydroxylase (Th) from molecularly defined dopamine neuron populations in mice. Broad deletion of Th in dopamine transporter-expressing neurons nearly abolished FAA, whereas restoration of Th in substantia nigra dopamine neurons rescued anticipatory locomotion. Surprisingly, deletion of Th from several large dopamine neuron populations had little effect on FAA. In contrast, deletion using Calb1Cre, targeting only [~]25% of substantia nigra dopamine neurons, produced a profound FAA deficit. Notably, these mice exhibited a profound loss of anticipatory locomotion while retaining substantial anticipatory food-seeking behavior. These findings identify a small Calbindin1+ dopamine population required for anticipatory locomotion and demonstrate that distinct behavioral components of food anticipation can be genetically dissociated.

neuroscience↗

bioRxiv2026

Cancer-derived Extracellular Vesicles for Targeted Delivery of EGFRvIII siRNA to Glioblastoma, Comparison of siRNA Loading Methods and Efficiency

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

pharmacology and toxicology↗

bioRxiv2026

A Knock-in Ntsr1-Flp Driver Enables Intersectional and Systemic Targeting of Heterogeneous Midbrain Dopamine Circuits

Precise genetic access to molecularly defined neuronal subpopulations is essential for dissecting circuit heterogeneity. We developed and validated a knock-in neurotensin receptor 1 (Ntsr1)Flpmouse line for intersectional targeting of Ntsr1-expressing neurons. Following delivery of Flp-dependent adeno-associated viral (AAV) reporters, robust recombination was observed throughout the the midbrain and several other brain regions. A subset of Ntsr1+ neurons in the substantia nigra and ventral tegmental area lacked dopaminergic markers, indicating that midbrain Ntsr1 populations comprise both dopaminergic and non-dopaminergic neurons. Systemic delivery of a Cre and Flp-dependent reporter in complementary dual-recombinase configurations revealed configuration-dependent differences in dopaminergic targeting. Cis-gene controls (DatCre;DatFlp) defined the maximal dopaminergic targeting ceiling and helped distinguish true non-dopaminergic targeting from recombinase-dependent off-target labeling. Finally, a dual-recombinase-dependent taCaspase-3 construct enabled selective ablation of midbrain dopamine neurons in vivo, establishing Ntsr1Flp as a versatile driver for scalable Boolean targeting.

neuroscience↗

bioRxiv2025

Metabolic reprogramming and altered ATP content impair neuroprotective functions of microglia in β-glucocerebrosidase deficiency models

Mutations in the GBA gene, which reduce {beta}-glucocerebrosidase (GCase) activity, represent the most significant genetic risk factor for Parkinsons disease (PD). Decreased GCase activity has also been observed in sporadic PD cases, supporting a broader role for GCase in the poorly understood mechanisms underlying PD etiopathogenesis. While most studies on the relationship between GBA mutations and PD have focused on neurons, evidence suggests that PD pathology promoted by GCase deficiency involves other cell types and, in particular, interactions between neuronal and glial cells. Here, we identify microglia as primary players undergoing significant alterations at early stages of the pathological processes triggered by a GCase impairment. Using both pharmacological and genetic mouse models of GCase deficiency, we observed microglial morphological, transcriptional and metabolic changes. Interestingly, these changes were associated with a cell-specific, significant reduction of microglial ATP levels. When microglial ATP depletion was reproduced in an in vitro system of co-cultured microglial and neuronal cells, the neuroprotective properties of microglia were compromised and neuronal susceptibility to oxidative stress was enhanced. These findings underscore the role of microglia in PD pathogenesis and point to a pathogenetic mechanism by which microglial metabolic disturbances leading to ATP depletion enhance neuronal vulnerability to injury and neurodegeneration. This mechanism could be targeted for therapeutic intervention aimed at mitigating PD risk and counteracting the development of PD pathology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/656111v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@dfd3d7org.highwire.dtl.DTLVardef@ccf2c2org.highwire.dtl.DTLVardef@155d241org.highwire.dtl.DTLVardef@15ed00a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Actin polymerization by WASp limits nuclear envelope rupture and inflammation in macrophages

Mutations in the immune-specific actin regulator WASp induce a proinflammatory state in myeloid cells, whose underlying causes remain poorly defined. Here, we applied microfabricated tools that mimic tissue mechanical forces to explore the role of WASp in connecting mechano-sensing to the activation of inflammatory responses in macrophages. We show that WASp-deficient macrophages carry alterations in nuclear structure and undergo increased blebbing and nuclear rupture when exposed to mechanical confinement. High-resolution imaging indicates that WASp drives the formation of protective perinuclear actin structures in response to confinement. Functionally, a proinflammatory gene signature linked to nuclear envelope rupture is preferentially active in confined WASp null macrophages, which partially depends on the cGAS-STING pathway of cytosolic DNA sensing. Analysis of transcriptional datasets of human and mouse tissue macrophages confirmed elevated inflammatory activation in WASp null cells. Together, these data uncover that WASp restricts pro-inflammatory activation of macrophages by preserving nuclear integrity in confined environments, providing novel clues to understand inflammatory activation in Wiskott-Aldrich Syndrome.

immunology↗

bioRxiv2024

Molecular dynamics analysis of axonal membrane during traumatic brain injury

Diffuse axonal injury (DAI), characterized by widespread damage to axons throughout the brain, represents one of the most devastating and difficult-to-treat forms of traumatic brain injury. Different theories exist about the mechanism of DAI, among which one hypothesis states that membrane poration of the axons initiates DAI. To investigate the hypothesis, molecular models of axonal membranes, incorporating 25 different lipids distributed asymmetrically in the leaflets, were developed using a coarse-grain description and simulated using molecular dynamics techniques. Different protein concentrations were embedded inside the lipid bilayer to describe the different sub-cellular parts in myelinated and unmyelinated axons. The models were investigated in equilibration and under deformation to characterize the structural and mechanical properties of the membranes, and comparisons were made with other subcellular parts, particularly myelin. Employing a bottom-top approach, the results were coupled with a finite element model representing the axon at the cell level. The results indicate that pore formation in the node-of-Ranvier occurs at a lower rupture strain compared to other axolemma parts, whereas myelin poration exhibits the highest rupture strains among the investigated models. The observed rupture strain for the node-of-Ranvier aligns with experimental studies, indicating a threshold for injury at axonal strains exceeding 10 - 13%epending on the strain rate. The results indicate that the hypothesis suggesting mechanoporation triggers axonal injury cannot be dismissed, as this phenomenon occurs within the threshold of axonal injury. HighlightsO_LIDeveloping a realistic molecular model of axolemma based on experimental data about its lipid composition C_LIO_LIInvestigating how lipid composition and protein concentration affect the membranes structural and mechanical properties C_LIO_LIIdentifying the most vulnerable regions of the axonal membrane C_LI

bioengineering↗

bioRxiv2023

Embryonic lymphocytes contribute to a genetic form of autoimmune inflammation

Omenn Syndrome (OS) is a rare hematological disorder, caused by hypomorphic mutations in genes involved in B-/T-cell receptor (BCR/TCR) rearrangement that result in impaired lymphocyte development and immunodeficiency. Notwithstanding, few T-cell clones enriched in self-reactive specificities expand in peripheral tissues, where they trigger severe inflammation and autoimmune reactions. Interestingly, residual OS lymphocytes display characteristics proper of embryonic lymphocytes that emerge before, and independently from, hematopoietic stem cells (HSCs). This prompted us to hypothesize whether OS autoreactive T-cells are generated in the embryo independently from HSCs. Here we show that in the Rag2R229Q/R229Q OS mouse model, embryonic but not adult bone marrow-derived hematopoietic progenitors can generate T-cells. T-lymphopoiesis can be rescued in adult OS blood progenitors via their Lin28-mediated reprogramming to an embryonic-like state. Remarkably, when transplanted in immunodeficient mice, embryonic-like OS progenitors trigger tissue morphological alterations and inflammation in the large intestine of the recipients, recapitulating the typical OS inflammatory phenotype. Our study describes the previously unappreciated contribution of embryonic progenitors to the pool of autoreactive infilitrating T-cells, providing a novel platform for both the detailed study of human autoimmune disorders and the design of more targeted therapies.

developmental biology↗

bioRxiv2023

Total RNA sequencing reveals gene expression and microbial alterations shared by oral pre-malignant lesions and cancer.

Head and neck cancers are a complex malignancy comprising multiple anatomical sites, with cancer of the oral cavity ranking among the deadliest and most disfiguring cancers globally. Oral cancer (OC) constitutes a subset of head and neck cancer cases, presenting primarily as tobacco-and alcohol-associated oral squamous cell carcinoma (OSCC), with a 5-year survival rate of [~]65%, partly due to the lack of early detection and effective treatments. OSCC arises from premalignant lesions (PMLs) in the oral cavity through a multi-step series of clinical and histopathological stages, including varying degrees of epithelial dysplasia. To gain insights into the molecular mechanisms associated with the progression of PMLs to OSCC, we profiled the whole transcriptome of 66 human PMLs comprising leukoplakia with dysplasia and hyperkeratosis non-reactive (HkNR) pathologies, alongside healthy controls and OSCC. Our data revealed that PMLs were enriched in gene signatures associated with cellular plasticity, such as partial EMT (p-EMT) phenotypes, and with immune response. Integrated analyses of the host transcriptome and microbiome further highlighted a significant association between differential microbial abundance and PML pathway activity, suggesting a contribution of the oral microbiome towards PML evolution to OSCC. Collectively, this study reveals molecular processes associated with PML progression that may help early diagnosis and disease interception at an early stage. AUTHOR SUMMARYPatients harboring oral premalignant lesions (PMLs) have an increased risk of developing oral squamous cell carcinoma (OSCC), but the underlying mechanisms driving transformation of PMLs to OSCC remain poorly understood. In this study, Khan et al., analyzed a newly generated dataset of gene expression and microbial profiles of oral tissues from patients diagnosed with PMLs from differing histopathological groups, including hyperkeratosis not reactive (HkNR) and dysplasia, comparing these profiles with OSCC and normal oral mucosa. Significant similarities between PMLs and OSCC were observed, with PMLs manifesting several cancer hallmarks, including oncogenic and immune pathways. The study also demonstrates associations between the abundance of multiple microbial species and PML groups, suggesting a potential contribution of the oral microbiome to the early stages of OSCC development. The study offers insights into the nature of the molecular, cellular and microbial heterogeneity of oral PMLs and suggests that molecular and clinical refinement of PMLs may provide opportunities for early disease detection and interception. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/534064v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@86cf7org.highwire.dtl.DTLVardef@129e6d1org.highwire.dtl.DTLVardef@1f0ae31org.highwire.dtl.DTLVardef@3c4d67_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

Sex-specific microglial responses to glucocerebrosidase inhibition: relevance to GBA1-linked Parkinson disease

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we analysed the dynamic changes in shape and motility occurring in primary mouse microglia following pro-inflammatory or neurotoxic insults, thus finding sex-specific responses of microglial subpopulations. Male microglia exhibited a pro-inflammatory phenotype, whereas female microglia showed enhanced neuroprotective capabilities associated with the activation of Nrf2 detoxification pathway in neurons. The sex difference in neuroprotective functions is lost by inhibition of glucocerebrosidase, the product of the GBA1 gene, mutations of which are the major risk factor for Parkinsons disease (PD). This finding is consistent with the increased risk of PD observed in female carriers of GBA1 mutation, when compared with wild type population, suggesting a role for microglial functionality in the etiopathogenesis of PD-GBA1.

neuroscience↗

bioRxiv2022

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and Synthetic Biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed "Super-Resolution Imaging" techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have however not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform Single-Molecule Localization Microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photo-convertible fluorescent protein mEos3.2, enabling Photo-Activated Localization Microscopy (PALM) in most Mycoplasma species. We also describe the application of direct STochastic Optical Reconstruction Microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development SMLM strategies to study these organisms in the future. ImportanceMycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small size of these bacteria, well below the micron, limits the usefulness of traditional fluorescence imaging methods as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art "Super-Resolution Microscopy" techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photo-convertible fluorescent protein; and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nanometers. Our work paves the way toward a better understanding of mycoplasmas biology through imaging of subcellular structures at the nanometer scale.

microbiology↗

bioRxiv2021

Inhibition of microglial GBA hampers the microglia-mediated anti-oxidant and protective response in neurons

Homozygotic mutations in the GBA gene cause Gauchers disease, moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinsons disease. In homozygosis, these mutations impair the activity of {beta}-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gauchers disease. In the brain, most of the pathological effects caused by GBA mutations have been attributed to the {beta}-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. Here, we applied the bioluminescence imaging technology, immunohistochemical and gene expression analysis to investigate the consequences of microglial {beta}-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia co-cultures and in cell lines. Our data demonstrate the existence of a novel mechanism by which microglia sustain the antioxidant/detoxifying response mediated by the nuclear factor erythroid 2-related factor 2 in neurons. The central role played by microglia in this neuronal response in vivo was proven by pharmacological depletion of the lineage in the brain, while co-cultures experiments provided insight on the nature of this cell-to-cell communication showing that this mechanism requires a direct microglia-to-neuron contact supported by functional actin structures. Pharmacological inhibition of microglial {beta}-glucocerebrosidase was proven to induce morphological changes, turn on an anti-inflammatory/repairing pathway and hinder the microglia ability to activate the anti-oxidant/detoxifying response, thus increasing the neuronal susceptibility to neurotoxins. Altogether, our data suggest that microglial {beta}-glucocerebrosidase inhibition impairs microglia-to-neuron communication increasing the sensitivity of neurons to oxidative or toxic insults, thus providing a possible mechanism for the increased risk of neurodegeneration observed in carriers of GBA mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/427380v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@15c2c3corg.highwire.dtl.DTLVardef@5c4e34org.highwire.dtl.DTLVardef@12718dorg.highwire.dtl.DTLVardef@dcfeca_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefMicroglia, through actin-dependent structures, contact neurons and induce a detoxification response by increasing the NFE2L2 signalling pathway. Inhibition of GCase activity by CBE treatment produces a morpho-functional change in microglia cells hampering the neuroprotective microglia-neuron communication thus inducing a phenotype in dopaminergic neurons characterized by increased susceptibility to oxidative stress or toxic insults.

neuroscience↗

bioRxiv2020

Type 1 dopamine receptor (D1R)-independent circadian food anticipatory activity in mice

Circadian rhythms are entrained by light and influenced by non-photic stimuli, such as feeding. The activity preceding scheduled mealtimes, food anticipatory activity (FAA), is elicited in rodents fed a limited amount at scheduled times. FAA is thought to be the output of an unidentified food entrained oscillator. Previous studies, using gene deletion and receptor pharmacology, implicated dopamine type receptor 1 (D1R) signaling in the dorsal striatum as necessary for FAA in mice. To further understand the role of D1R in promoting FAA, we utilized the Cre-lox system to create cell type-specific deletions of D1R. We were unsuccessful in obtaining conditional deletion of D1R when using transgenically driven D1R-Cre. We then created a conditional deletion of D1R in GABA neurons using Vgat-ires-Cre line, which had attenuated FAA, but the amount was higher than expected based on prior results using a constitutive knockout of D1R, D1R KODrago. This result prompted us to re-test the original D1R KODrago line, which expressed less FAA than controls, but only moderately so. To determine if genetic drift had diminished the effect of D1R deletion on FAA, we re-established the D1R KODrago knockout line from cryopreserved samples. The reestablished D1R KODrago-cryo had a clear impairment of FAA compared to controls, but still developed increased activity preceding mealtime across the 4 weeks of timed feeding. Finally, we tested a different deletion allele of D1R created by the Knockout Mouse Project. This line of D1R KOKOMP mice had a significant impairment in the acquisition of FAA, but eventually reached similar levels of premeal activity compared to controls after 4 weeks of timed feeding. Taken together, our results suggest that D1R signaling promotes FAA, but other dopamine receptors likely contribute to FAA given that mice lacking the D1 receptor still retain some FAA.

neuroscience↗

bioRxiv2020

Antibody-based delivery of Interleukin-9 to neovascular structures: therapeutic evaluation in cancer and arthritis

Interleukin-9 (IL9) is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells (ILC2s), which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, IL9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression and characterization of three fusion proteins based on murine IL9 and the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. IL9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the IL9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of IL9 was retained when the payload was fused to antibodies. However, the targeted delivery of IL9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26 and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver IL9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.

bioengineering↗

bioRxiv2020

Sortase-mediated site-specific modification of interleukin-2 for the generation of a tumor targeting acetazolamide-cytokine conjugate

1.Small ligands specific to tumor-associated antigens can be used as alternatives to antibodies for the delivery of small payloads such as radionuclides, cytotoxic drugs and fluorophores. Their use as delivery moiety of bioactive proteins like cytokines remains largely unexplored. Here, we describe the preparation and in vivo characterization of the first small molecule-cytokine conjugate targeting carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma and hypoxia. Site-specific conjugation between interleukin-2 and acetazolamide was obtained by Sortase A-mediated transpeptidation. Binding of the conjugate to the cognate CAIX antigen was confirmed by surface plasmon resonance. The in vivo targeting of structures expressing carbonic anhydrase IX was assessed by biodistribution experiments in tumor bearing mice. Optimization of manufacturability and tumor targeting performance of acetazolamide-cytokine products will be required in order to enable industrial applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/211870v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@fb64d8org.highwire.dtl.DTLVardef@99aebeorg.highwire.dtl.DTLVardef@19f74acorg.highwire.dtl.DTLVardef@1a4de93_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Immunotherapy with immunocytokines and PD-1 blockade enhances the anticancer activity of small molecule-drug conjugates targeting carbonic anhydrase IX

Small molecule-drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article we describe novel combination therapies consisting of anti-Carbonic Anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody-cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively-spliced EDB domain of fibronectin containing the murine interleukin-12 moiety, was active also against large established tumors. Analysis of the microscopic structures of healthy organs performed three months after tumor eradication confirmed absence of pathological abnormalities in the healthy kidney, liver, lung, stomach and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on small molecule-drug conjugates and immunotherapy for the treatment of renal cell carcinoma and of hypoxic tumors.

cancer biology↗