Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping.

Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors.

Adenovirus-α-Defensin Complexes Induce NLRP3-Associated Maturation of Human Phagocytes via Toll-Like Receptor 4 Engagement.

Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 h is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences direct and indirect innate immune responses to a range of pathogens, impacts the response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids and redirects HAdV-C5, -D26, and -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and interleukin 1 beta (IL-1ß) release. Surprisingly, IL-1ß release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV vaccine immunogenicity and may provide pathways to extend the efficacy. IMPORTANCE This study examines the interactions between danger-associated molecular patterns and human adenoviruses, and their impact on vaccines. HAdVs and HNP-1 can interact, and these interactions will modify the response of antigen-presenting cells, which will influence vaccine efficacy.

What is the risk of a deadly adenovirus pandemic?

Many of us had refresher courses in virology, immunology, and epidemiology in 2020, and we were reminded of the fact that Homo sapiens, the wiliest predator on the planet, has been hunting everything that moves for millennia. These repeated interspecies contacts inherently lead to recurrent zoonosis (nonhuman to human) and anthroponosis (human to nonhuman). Given the accelerating changes in our ecosystems since the neolithic revolution, it was not surprising to see a virus that spreads via aerosolization and liquid droplets cause a pandemic in a few months. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic begs the question-which viruses could cause a global threat? In this Opinion, the characteristics that make adenoviruses a risk, which include efficient intra- and interspecies transmission, thermostable particles, persistent/latent infections in diverse hosts, and the ability to readily recombine and escape herd immunity, are discussed.

Adenovirus receptors on antigen-presenting cells of the skin.

Skin, the largest human organ, is part of the first line of physical and immunological defense against many pathogens. Understanding how skin antigen-presenting cells (APCs) respond to viruses or virus-based vaccines is crucial to develop antiviral pharmaceutics, and efficient and safe vaccines. Here, we discuss the way resident and recruited skin APCs engage adenoviruses and the impact on innate immune responses.

Adenovirus-based vaccines-a platform for pandemic preparedness against emerging viral pathogens.

Zoonotic viruses continually pose a pandemic threat. Infection of humans with viruses for which we typically have little or no prior immunity can result in epidemics with high morbidity and mortality. These epidemics can have public health and economic impact and can exacerbate civil unrest or political instability. Changes in human behavior in the past few decades-increased global travel, farming intensification, the exotic animal trade, and the impact of global warming on animal migratory patterns, habitats, and ecosystems-contribute to the increased frequency of cross-species transmission events. Investing in the pre-clinical advancement of vaccine candidates against diverse emerging viral threats is crucial for pandemic preparedness. Replication-defective adenoviral (Ad) vectors have demonstrated their utility as an outbreak-responsive vaccine platform during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Ad vectors are easy to engineer; are amenable to rapid, inexpensive manufacturing; are relatively safe and immunogenic in humans; and, importantly, do not require specialized cold-chain storage, making them an ideal platform for equitable global distribution or stockpiling. In this review, we discuss the progress in applying Ad-based vaccines against emerging viruses and summarize their global safety profile, as reflected by their widespread geographic use during the SARS-CoV-2 pandemic.

Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.

Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

Humoral immune response to adenovirus induce tolerogenic bystander dendritic cells that promote generation of regulatory T cells.

Following repeated encounters with adenoviruses most of us develop robust humoral and cellular immune responses that are thought to act together to combat ongoing and subsequent infections. Yet in spite of robust immune responses, adenoviruses establish subclinical persistent infections that can last for decades. While adenovirus persistence pose minimal risk in B-cell compromised individuals, if T-cell immunity is severely compromised reactivation of latent adenoviruses can be life threatening. This dichotomy led us to ask how anti-adenovirus antibodies influence adenovirus T-cell immunity. Using primary human blood cells, transcriptome and secretome profiling, and pharmacological, biochemical, genetic, molecular, and cell biological approaches, we initially found that healthy adults harbor adenovirus-specific regulatory T cells (Tregs). As peripherally induced Tregs are generated by tolerogenic dendritic cells (DCs), we then addressed how tolerogenic DCs could be created. Here, we demonstrate that DCs that take up immunoglobulin-complexed (IC)-adenoviruses create an environment that causes bystander DCs to become tolerogenic. These adenovirus antigen loaded tolerogenic DCs can drive naïve T cells to mature into adenovirus-specific Tregs. Our study reveals a mechanism by which an antiviral humoral responses could, counterintuitively, favor virus persistence.

Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells.

Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1ß and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs.

Coxsackievirus Adenovirus Receptor Loss Impairs Adult Neurogenesis, Synapse Content, and Hippocampus Plasticity.

UNLABELLED: Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT: This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

Human coagulation factor X-adenovirus type 5 complexes poorly stimulate an innate immune response in human mononuclear phagocytes.

UNLABELLED: One of the first lines of host defense against many viruses in vertebrates is the innate immune system, which detects pathogen-associated molecular patterns (PAMPs) using pathogen recognition receptors (PRR). The dynamic interactions between pathogens and hosts create, in some cases, species-specific relationships. Recently, it was shown that murine factor X (mFX)-armored human adenovirus (HAd) stimulated a mFX-Toll-like receptor 4 (TLR4)-associated response in mouse macrophages in vitro and in vivo. Given the importance of studies using animals to better understand host-pathogen interactions, we asked if human FX (hFX)-armored HAd type 5 (HAd5) was capable of activating innate immune sensors in primary human mononuclear phagocytes. To this end, we assayed human mononuclear phagocytes for their ability to be stimulated by hFX-armored HAd5 via a TLR/NF-κB pathway, in particular, a TLR4 pathway. In our hands, we found no significant interaction, activation, or maturation of human mononuclear phagocytes caused by the presence of hFX-armored HAd5. IMPORTANCE: Animals, and mice in particular, are often used as informative and powerful surrogates for how pathogens interact with natural host systems. When possible, extended and targeted studies in the natural host can then be performed. Our data will help us understand the differences in preclinical testing in mice and clinical use in humans in order to improve treatment for HAd diseases and Ad vector effectiveness.

The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FK(CAV)), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FK(CAV) entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE: Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.

Disruption of the coxsackievirus and adenovirus receptor-homodimeric interaction triggers lipid microdomain- and dynamin-dependent endocytosis and lysosomal targeting.

The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail.

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain.

Severe deficiency in lysosomal ß-glucuronidase (ß-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in ß-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, ß-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

Toll-like receptor 4 - a multifunctional virus recognition receptor.

Since the initial description of Toll receptors in Drosophila and their mammalian counterparts Toll-like receptors (TLRs), numerous fundamental and applied studies have explored their crucial role as sensors of pathogen-associated molecular patterns (PAMPs). Among the ten human TLRs, TLR4 is particularly well known for its ability to detect lipopolysaccharides (LPS), a component of the Gram-negative bacterial cell wall. In addition to its archetypal functions, TLR4 is also a versatile virus sensor. This review provides a background on the discovery of TLR4 and how this knowledge laid a foundation for characterization of its diverse roles in antiviral responses, examined through genetic, biochemical, structural, and immunological approaches. These advances have led to a deeper understanding of the molecular functions that enable TLR4 to orchestrate multi-nodal control by professional antigen-presenting cells (APCs) to initiate appropriate and regulated antiviral immune responses.

The number of Toll-like receptor 9-agonist motifs in the adenovirus genome correlates with induction of dendritic cell maturation by adenovirus immune complexes.

Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.

3D cultures of human neural progenitor cells: dopaminergic differentiation and genetic modification. [corrected].

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety. Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10 days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson's disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo. Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson's disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.

Inhibition of noradrenergic signalling in rodent orbitofrontal cortex impairs the updating of goal-directed actions.

In a constantly changing environment, organisms must track the current relationship between actions and their specific consequences and use this information to guide decision-making. Such goal-directed behaviour relies on circuits involving cortical and subcortical structures. Notably, a functional heterogeneity exists within the medial prefrontal, insular, and orbitofrontal cortices (OFC) in rodents. The role of the latter in goal-directed behaviour has been debated, but recent data indicate that the ventral and lateral subregions of the OFC are needed to integrate changes in the relationships between actions and their outcomes. Neuromodulatory agents are also crucial components of prefrontal functions and behavioural flexibility might depend upon the noradrenergic modulation of the prefrontal cortex. Therefore, we assessed whether noradrenergic innervation of the OFC plays a role in updating action-outcome relationships in male rats. We used an identity-based reversal task and found that depletion or chemogenetic silencing of noradrenergic inputs within the OFC rendered rats unable to associate new outcomes with previously acquired actions. Silencing of noradrenergic inputs in the prelimbic cortex or depletion of dopaminergic inputs in the OFC did not reproduce this deficit. Together, our results suggest that noradrenergic projections to the OFC are required to update goal-directed actions.

Viral vector-mediated expression of NaV1.1, after seizure onset, reduces epilepsy in mice with Dravet syndrome.

Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally induced seizures, corrected background electrocorticographic activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof of concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.

A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galß1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid.