Neurotensin orchestrates valence assignment in the amygdala.

The ability to associate temporally segregated information and assign positive or negative valence to environmental cues is paramount for survival. Studies have shown that different projections from the basolateral amygdala (BLA) are potentiated following reward or punishment learning1-7. However, we do not yet understand how valence-specific information is routed to the BLA neurons with the appropriate downstream projections, nor do we understand how to reconcile the sub-second timescales of synaptic plasticity8-11 with the longer timescales separating the predictive cues from their outcomes. Here we demonstrate that neurotensin (NT)-expressing neurons in the paraventricular nucleus of the thalamus (PVT) projecting to the BLA (PVT-BLA:NT) mediate valence assignment by exerting NT concentration-dependent modulation in BLA during associative learning. We found that optogenetic activation of the PVT-BLA:NT projection promotes reward learning, whereas PVT-BLA projection-specific knockout of the NT gene (Nts) augments punishment learning. Using genetically encoded calcium and NT sensors, we further revealed that both calcium dynamics within the PVT-BLA:NT projection and NT concentrations in the BLA are enhanced after reward learning and reduced after punishment learning. Finally, we showed that CRISPR-mediated knockout of the Nts gene in the PVT-BLA pathway blunts BLA neural dynamics and attenuates the preference for active behavioural strategies to reward and punishment predictive cues. In sum, we have identified NT as a neuropeptide that signals valence in the BLA, and showed that NT is a critical neuromodulator that orchestrates positive and negative valence assignment in amygdala neurons by extending valence-specific plasticity to behaviourally relevant timescales.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics.

Sex-dependent neurobiological features of prenatal immune activation via TLR7.

Immune activation during pregnancy via infection or autoimmune disease is a risk factor for neuropsychiatric illness. Mouse models of prenatal immune activation often involve maternal administration of agents that activate toll-like receptors (TLRs), a class of pattern recognition receptors that initiate innate immune responses. Such studies have focused primarily on activating the TLR3 or TLR4 subtypes, to mimic immune responses to viral or bacterial infections, respectively. Here, we characterize the effects of prenatal activation of TLR7, which is implicated in the pathogenesis of autoimmune disease. Prenatal TLR7 activation via administration of the selective agonist imiquimod (5.0 mg/kg) induces a phenotype in offspring characterized by reduced anxiety-like behavior, fragmented social behavior, and altered ultrasonic vocalization patterns at 6-12 weeks of age. The characteristics of this phenotype are readily distinguishable from-and in some ways opposite to-those seen following prenatal activation of TLR3 and/or TLR4. Prenatal TLR7-activated mice have normal baseline locomotor activity, but are hyperresponsive to stimuli including social partners, circadian cues, and gonadal hormone fluctuations. These alterations are accompanied by decreases in microglia density but increases in ramifications. RNA-sequencing of dorsal striatum, a region showing profound changes in microglial markers, indicates that prenatal TLR7 activation induces differential expression of hundreds of genes at 13 weeks of age, with virtually no overlap in differentially expressed genes between males and females. Our findings demonstrate that prenatal immune activation can promote a wide range of developmental trajectories, depending on the type and/or pattern of TLR activation and the sex of the offspring.

Glycan-binding profile of DC-like cells.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-"vector" it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1-3(Manα1-6)Manß1-4GlcNAcß1-4GlcNAcß bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1-3Galß (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Dendritic Cell Sensing of Hydrophobic Di- and Triacylated Lipopeptides Self-Assembled within Synthetic Virus-like Particles.

Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1+ early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker+ structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP+ DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14low/-CD16+CD83+ blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14low/-CD16+CD83+ subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 low/-CD16+CD83+ subpopulation was observed for GalNAcα1-2Galß (Adi), (Neu5Acα)3 and three mannose-reach glycans; 3) subpopulation of CD14low/-CD16+ cells preferentially bound 4'-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 low/-CD16+CD83+ cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan "vectors" and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on Adi and (Neu5Acα)3 as the most promising vectors.

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines.

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14 kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR: The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Thy1-expressing neurons in the basolateral amygdala may mediate fear inhibition.

Research has identified distinct neuronal circuits within the basolateral amygdala (BLA) that differentially mediate fear expression versus inhibition; however, molecular markers of these populations remain unknown. Here we examine whether optogenetic activation of a cellular subpopulation, which may correlate with the physiologically identified extinction neurons in the BLA, would differentially support fear conditioning versus fear inhibition/extinction. We first molecularly characterized Thy1-channelrhodopsin-2 (Thy1-ChR2-EYFP)-expressing neurons as a subpopulation of glutamatergic pyramidal neurons within the BLA. Optogenetic stimulation of these neurons inhibited a subpopulation of medial central amygdala neurons and shunted excitation from the lateral amygdala. Brief activation of these neurons during fear training disrupted later fear memory in male mice. Optogenetic activation during unreinforced stimulus exposure enhanced extinction retention, but had no effect on fear expression, locomotion, or open-field behavior. Together, these data suggest that the Thy1-expressing subpopulation of BLA pyramidal neurons provide an important molecular and pharmacological target for inhibiting fear and enhancing extinction and for furthering our understanding of the molecular mechanisms of fear processing.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR: This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Single nuclei transcriptomics in human and non-human primate striatum in opioid use disorder.

In brain, the striatum is a heterogenous region involved in reward and goal-directed behaviors. Striatal dysfunction is linked to psychiatric disorders, including opioid use disorder (OUD). Striatal subregions are divided based on neuroanatomy, each with unique roles in OUD. In OUD, the dorsal striatum is involved in altered reward processing, formation of habits, and development of negative affect during withdrawal. Using single nuclei RNA-sequencing, we identified both canonical (e.g., dopamine receptor subtype) and less abundant cell populations (e.g., interneurons) in human dorsal striatum. Pathways related to neurodegeneration, interferon response, and DNA damage were significantly enriched in striatal neurons of individuals with OUD. DNA damage markers were also elevated in striatal neurons of opioid-exposed rhesus macaques. Sex-specific molecular differences in glial cell subtypes associated with chronic stress were found in OUD, particularly female individuals. Together, we describe different cell types in human dorsal striatum and identify cell type-specific alterations in OUD.

SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration.

High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. Here we show that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1ß release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in male mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1ß release, contributing to an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of male and female postmortem human brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing mechanistic insight into the biology of neuroinflammation.

Chicken cells sense influenza A virus infection through MDA5 and CARDIF signaling involving LGP2.

Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-ß (chIFN-ß) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-ß levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-ß promoter, the silencing of endogenous chLGP2 reduced chIFN-ß induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.

Alginate-coated chitosan nanogel capacity to modulate the effect of TLR ligands on blood dendritic cells.

Biodegradable nanoparticles have been employed for vaccine delivery, frequently admixed with adjuvants. Surprisingly, there is little information on their modulation of immune responses, speculated to be negligible. We analyzed the immunomodulatory capacity of alginate-coated chitosan nanogels (Ng), on porcine and human blood dendritic cells (DCs), when applied with defined adjuvants targeting different DC subpopulations. DC maturation, cytokine production and cell migration were assessed. Ng differentially influenced the immunomodulatory characteristics of individual Toll-like receptor (TLR) ligands: Pam3Cys-SK4-induced IL-1ß was enhanced; CpG-oligodeoxynucleotides (CpG-ODN)-induced IFN-α, IL-6 and TNFα were impaired; CpG-ODN-induced CD86 and CCR7, and cell migration, were diminished-plasmacytoid DCs (pDCs) were particularly sensitive. Therein, the Ng influence on DC endocytosis of the TLR ligands was apparently a major contributory element. This demonstrates the importance of predefining the interplay between delivery vehicles and admixed immunostimulatory moieties, for ensuring appropriate immune activation and efficacious combinations. FROM THE CLINICAL EDITOR: Biodegradable nanoparticles have been utilized in vaccine delivery; however, there is little information available on their immunomodulatory properties, which are thought to be negligible. This study clearly demonstrates that nanogels do influence the developing immune response, which needs to be taken into consideration when utilizing these otherwise very efficacious vaccine delivery approaches.

Differential effects of the stress peptides PACAP and CRF on sleep architecture in mice.

Stress produces profound effects on behavior, including persistent alterations in sleep patterns. Here we examined the effects of two prototypical stress peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing factor (CRF), on sleep architecture and other translationally-relevant endpoints. Male and female mice were implanted with subcutaneous transmitters enabling continuous measurement of electroencephalography (EEG) and electromyography (EMG), as well as body temperature and locomotor activity, without tethering that restricts free movement, body posture, or head orientation during sleep. At baseline, females spent more time awake (AW) and less time in slow wave sleep (SWS) than males. Mice then received intracerebral infusions of PACAP or CRF at doses producing equivalent increases in anxiety-like behavior. The effects of PACAP on sleep architecture were similar in both sexes and resembled those reported in male mice after chronic stress exposure. Compared to vehicle infusions, PACAP infusions decreased time in AW, increased time in SWS, and increased rapid eye movement sleep (REM) time and bouts on the day following treatment. In addition, PACAP effects on REM time remained detectable a week after treatment. PACAP infusions also reduced body temperature and locomotor activity. Under the same experimental conditions, CRF infusions had minimal effects on sleep architecture in either sex, causing only transient increases in SWS during the dark phase, with no effects on temperature or activity. These findings suggest that PACAP and CRF have fundamentally different effects on sleep-related metrics, and provide new insights into the mechanisms by which stress disrupts sleep.

Single-Nucleus Transcriptome Profiling of Dorsolateral Prefrontal Cortex: Mechanistic Roles for Neuronal Gene Expression, Including the 17q21.31 Locus, in PTSD Stress Response.

OBJECTIVE: Multidisciplinary studies of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) implicate the dorsolateral prefrontal cortex (DLPFC) in disease risk and pathophysiology. Postmortem brain studies have relied on bulk-tissue RNA sequencing (RNA-seq), but single-cell RNA-seq is needed to dissect cell-type-specific mechanisms. The authors conducted the first single-nucleus RNA-seq postmortem brain study in PTSD to elucidate disease transcriptomic pathology with cell-type-specific resolution. METHOD: Profiling of 32 DLPFC samples from 11 individuals with PTSD, 10 with MDD, and 11 control subjects was conducted (∼415K nuclei; >13K cells per sample). A replication sample included 15 DLPFC samples (∼160K nuclei; >11K cells per sample). RESULTS: Differential gene expression analyses identified significant single-nucleus RNA-seq differentially expressed genes (snDEGs) in excitatory (EX) and inhibitory (IN) neurons and astrocytes, but not in other cell types or bulk tissue. MDD samples had more false discovery rate-corrected significant snDEGs, and PTSD samples had a greater replication rate. In EX and IN neurons, biological pathways that were differentially enriched in PTSD compared with MDD included glucocorticoid signaling. Furthermore, glucocorticoid signaling in induced pluripotent stem cell (iPSC)-derived cortical neurons demonstrated greater relevance in PTSD and opposite direction of regulation compared with MDD, especially in EX neurons. Many snDEGs were from the 17q21.31 locus and are particularly interesting given causal roles in disease pathogenesis and DLPFC-based neuroimaging (PTSD: ARL17B, LINC02210-CRHR1, and LRRC37A2; MDD: LRRC37A and LRP4), while others were regulated by glucocorticoids in iPSC-derived neurons (PTSD: SLC16A6, TAF1C; MDD: CDH3). CONCLUSIONS: The study findings point to cell-type-specific mechanisms of brain stress response in PTSD and MDD, highlighting the importance of examining cell-type-specific gene expression and indicating promising novel biomarkers and therapeutic targets.

SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration.

High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. We demonstrate that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1ß release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1ß release, initiating an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D (GSDMD)-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of postmortem brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing new mechanistic insight into the biology of neuroinflammation.

Blood levels of T-Cell Receptor Excision Circles (TRECs) provide an index of exposure to traumatic stress in mice and humans.

Exposure to stress triggers biological changes throughout the body. Accumulating evidence indicates that alterations in immune system function are associated with the development of stress-associated illnesses such as major depressive disorder and post-traumatic stress disorder, increasing interest in identifying immune markers that provide insight into mental health. Recombination events during T-cell receptor rearrangement and T-cell maturation in the thymus produce circular DNA fragments called T-cell receptor excision circles (TRECs) that can be utilized as indicators of thymic function and numbers of newly emigrating T-cells. Given data suggesting that stress affects thymus function, we examined whether blood levels of TRECs might serve as a quantitative peripheral index of cumulative stress exposure and its physiological correlates. We hypothesized that chronic stress exposure would compromise thymus function and produce corresponding decreases in levels of TRECs. In male mice, exposure to chronic social defeat stress (CSDS) produced thymic involution, adrenal hypertrophy, and decreased levels of TRECs in blood. Extending these studies to humans revealed robust inverse correlations between levels of circulating TRECs and childhood emotional and physical abuse. Cell-type specific analyses also revealed associations between TREC levels and blood cell composition, as well as cell-type specific methylation changes in CD4T + and CD8T + cells. Additionally, TREC levels correlated with epigenetic age acceleration, a common biomarker of stress exposure. Our findings demonstrate alignment between findings in mice and humans and suggest that blood-borne TRECs are a translationally-relevant biomarker that correlates with, and provides insight into, the cumulative physiological and immune-related impacts of stress exposure in mammals.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells.

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.