Modulation of cannabinoid receptor 2 alters neuroinflammation and reduces formation of alpha-synuclein aggregates in a rat model of nigral synucleinopathy.

Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to chronic neuroinflammation and neuronal loss in Parkinson's disease (PD). There is growing evidence that neuroinflammation accompanies and may promote progression of alpha-synuclein (Asyn)-induced nigral dopaminergic (DA) degeneration. From a therapeutic perspective, development of immunomodulatory strategies that dampen overproduction of pro-inflammatory cytokines from chronically activated immune cells and induce a pro-phagocytic phenotype is expected to promote Asyn removal and protect vulnerable neurons. Cannabinoid receptor-2 (CB2) is highly expressed on activated microglia and peripheral immune cells, is upregulated in the substantia nigra of individuals with PD and in mouse models of nigral degeneration. Furthermore, modulation of CB2 protects against rotenone-induced nigral degeneration; however, CB2 has not been pharmacologically and selectively targeted in an Asyn model of PD. Here, we report that 7 weeks of peripheral administration of CB2 inverse agonist SMM-189 reduced phosphorylated (pSer129) alpha-synuclein in the substantia nigra compared to vehicle treatment. Additionally, SMM-189 delayed Asyn-induced immune cell infiltration into the brain as determined by flow cytometry, increased CD68 protein expression, and elevated wound-healing-immune-mediator gene expression. Additionally, peripheral immune cells increased wound-healing non-classical monocytes and decreased pro-inflammatory classical monocytes. In vitro analysis of RAW264.7 macrophages treated with lipopolysaccharide (LPS) and SMM-189 revealed increased phagocytosis as measured by the uptake of fluorescence of pHrodo E. coli bioparticles. Together, results suggest that targeting CB2 with SMM-189 skews immune cell function toward a phagocytic phenotype and reduces toxic aggregated species of Asyn. Our novel findings demonstrate that CB2 may be a target to modulate inflammatory and immune responses in proteinopathies.

Effects of prenatal pesticide exposure on the fetal brain and placenta transcriptomes in a rodent model.

Organophosphate and pyrethroid pesticides are among the most extensively used insecticides worldwide. Prenatal exposures to both classes of pesticides have been linked to a wide range of neurobehavioral deficits in the offspring. The placenta is a neuroendocrine organ and the crucial regulator of the intrauterine environment; early-life toxicant exposures could impact neurobehavior by disrupting placental processes. Female C57BL/6 J mice were exposed via oral gavage to an organophosphate, chlorpyrifos (CPF) at 5 mg/kg, a pyrethroid, deltamethrin (DM), at 3 mg/kg, or vehicle only control (CTL). Exposure began two weeks before breeding and continued every three days until euthanasia at gestational day 17. The transcriptomes of fetal brain (CTL n = 18, CPF n = 6, DM n = 8) and placenta (CTL n = 19, CPF n = 16, DM n = 12) were obtained through RNA sequencing, and resulting data was evaluated using weighted gene co-expression networks, differential expression, and pathway analyses. Fourteen brain gene co-expression modules were identified; CPF exposure disrupted the module related to ribosome and oxidative phosphorylation, whereas DM disrupted the modules related to extracellular matrix and calcium signaling. In the placenta, network analyses revealed 12 gene co-expression modules. While CPF exposure disrupted modules related to endocytosis, Notch and Mapk signaling, DM exposure dysregulated modules linked to spliceosome, lysosome and Mapk signaling pathways. Overall, in both tissues, CPF exposure impacted oxidative phosphorylation, while DM was linked to genes involved in spliceosome and cell cycle. The transcription factor Max involved in cell proliferation was overexpressed by both pesticides in both tissues. In summary, gestational exposure to two different classes of pesticide can induce similar pathway-level transcriptome changes in the placenta and the brain; further studies should investigate if these changes are linked to neurobehavioral impairments.

Soluble TNF mediates amyloid-independent, diet-induced alterations to immune and neuronal functions in an Alzheimer's disease mouse model.

Introduction: Increasing evidence indicates that neurodegenerative diseases, including Alzheimer's disease (AD), are a product of gene-by-environment interplay. The immune system is a major contributor mediating these interactions. Signaling between peripheral immune cells and those within the microvasculature and meninges of the central nervous system (CNS), at the blood-brain barrier, and in the gut likely plays an important role in AD. The cytokine tumor necrosis factor (TNF) is elevated in AD patients, regulates brain and gut barrier permeability, and is produced by central and peripheral immune cells. Our group previously reported that soluble TNF (sTNF) modulates cytokine and chemokine cascades that regulate peripheral immune cell traffic to the brain in young 5xFAD female mice, and in separate studies that a diet high in fat and sugar (HFHS) dysregulates signaling pathways that trigger sTNF-dependent immune and metabolic responses that can result in metabolic syndrome, which is a risk factor for AD. We hypothesized that sTNF is a key mediator of peripheral immune cell contributions to gene-by-environment interactions to AD-like pathology, metabolic dysfunction, and diet-induced gut dysbiosis. Methods: Female 5xFAD mice were subjected to HFHS diet for 2 months and then given XPro1595 to inhibit sTNF for the last month or saline vehicle. We quantified immune cell profiles by multi-color flow cytometry on cells isolated from brain and blood; metabolic, immune, and inflammatory mRNA and protein marker biochemical and immunhistological analyses, gut microbiome, and electrophysiology in brain slices were also performed. Results: Here, we show that selective inhibition of sTNF signaling via the biologic XPro1595 modulates the effects of an HFHS diet in 5xFAD mice on peripheral and central immune profiles including CNS-associated CD8+ T cells, the composition of gut microbiota, and long-term potentiation deficits. Discussion: Obesogenic diet induces immune and neuronal dysfunction in 5xFAD mice and sTNF inhibition mitigates its effects. A clinical trial in subjects at risk for AD due to genetic predisposition and underlying inflammation associated with peripheral inflammatory co-morbidities will be needed to investigate the extent to which these findings translate to the clinic.

Correlated evolution between orb weaver glue droplets and supporting fibres maintains their distinct biomechanical roles in adhesion.

Orb weaving spiders employ a 'silken toolkit' to accomplish a range of tasks, including retaining prey that strike their webs. This is accomplished by a viscous capture spiral thread that features tiny glue droplets, supported by a pair of elastic flagelliform fibres. Each droplet contains a glycoprotein core responsible for adhesion. However, prey retention relies on the integrated performance of multiple glue droplets and their supporting fibres, with previous studies demonstrating that a suspension bridge forms, whose biomechanics sum the adhesive forces of multiple droplets while dissipating the energy of the struggling insect. While the interdependence of the droplet's glycoprotein and flagelliform fibres for functional adhesion is acknowledged, there has been no direct test of this hypothesized linkage between the material properties of each component. Spider mass, which differs greatly across orb weaving species, also has the potential to affect flagelliform fibre and glycoprotein material properties. Previous studies have linked spider mass to capture thread performance but have not examined the relationship between spider mass and thread material properties. We extend earlier studies to examine these relationships in 16 orb weaving species using phylogenetic generalized least squares. This analysis revealed that glycoprotein stiffness (elastic modulus) was correlated with flagelliform fibre stiffness, and that spider mass was related to the glycoprotein volume, flagelliform fibre cross-sectional area and droplets per unit thread length. By shaping the elastic moduli of glycoprotein adhesive and flagelliform fibres, natural selection has maintained the biomechanical integration of this adhesive system.

Hospital sink traps as a potential source of the emerging multidrug-resistant pathogen Cupriavidus pauculus: characterization and draft genome sequence of strain MF1.

Introduction. Cupriavidus pauculus is historically found in soil and water but has more recently been reported to cause human infection and death. Hospital sink traps can serve as a niche for bacterial persistence and a platform for horizontal gene transfer, with evidence of dissemination of pathogens in hospital plumbing systems driving nosocomial infection.Gap Statement. This paper presents the first C. pauculus strain isolated from a hospital sink trap. There are only six genome assemblies available on NCBI for C. pauculus; two of these are PacBio/Illumina hybrids. This paper presents the first ONT/Illumina hybrid assembly, with five contigs. The other assemblies available consist of 37, 38, 111 and 227 contigs. This paper also presents data on biofilm formation and lethal dose in Galleria mellonella; there is little published information describing these aspects of virulence.Aim. The aims were to identify the isolate found in a hospital sink trap, characterize its genome, and assess whether it could pose a risk to human health.Methodology. The genome was sequenced, and a hybrid assembly of short and long reads produced. Antimicrobial susceptibility was determined by the broth microdilution method. Virulence was assessed by measuring in vitro biofilm formation compared to Pseudomonas aeruginosa and in vivo lethality in Galleria mellonella larvae.Results. The isolate was confirmed to be a strain of C. pauculus, with a 6.8 Mb genome consisting of 6468 coding sequences and an overall G+C content of 63.9 mol%. The genome was found to contain 12 antibiotic resistance genes, 8 virulence factor genes and 33 metal resistance genes. The isolate can be categorized as resistant to meropenem, amoxicillin, amikacin, gentamicin and colistin, but susceptible to cefotaxime, cefepime, imipenem and ciprofloxacin. Clear biofilm formation was seen in all conditions over 72 h and exceeded that of P. aeruginosa when measured at 37 °C in R2A broth. Lethality in G. mellonella larvae over 48 h was relatively low.Conclusion. The appearance of a multidrug-resistant strain of C. pauculus in a known pathogen reservoir within a clinical setting should be considered concerning. Further work should be completed to compare biofilm formation and in vivo virulence between clinical and environmental strains, to determine how easily environmental strains may establish human infection. Infection control teams and clinicians should be aware of the emerging nature of this pathogen and further work is needed to minimize the impact of contaminated hospital plumbing systems on patient outcomes.

Progranulin loss results in sex-dependent dysregulation of the peripheral and central immune system.

Introduction: Progranulin (PGRN) is a secreted glycoprotein, the expression of which is linked to several neurodegenerative diseases. Although its specific function is still unclear, several studies have linked it with lysosomal functions and immune system regulation. Here, we have explored the role of PGRN in peripheral and central immune system homeostasis by investigating the consequences of PGRN deficiency on adaptive and innate immune cell populations. Methods: First, we used gene co-expression network analysis of published data to test the hypothesis that Grn has a critical role in regulating the activation status of immune cell populations in both central and peripheral compartments. To investigate the extent to which PGRN-deficiency resulted in immune dysregulation, we performed deep immunophenotyping by flow cytometry of 19-24-month old male and female Grn-deficient mice (PGRN KO) and littermate Grn-sufficient controls (WT). Results: Male PGRN KO mice exhibited a lower abundance of microglial cells with higher MHC-II expression, increased CD44 expression on monocytes in the brain, and more CNS-associated CD8+ T cells compared to WT mice. Furthermore, we observed an increase in CD44 on CD8+ T cells in the peripheral blood. Female PGRN KO mice also had fewer microglia compared to WT mice, and we also observed reduced expression of MHC-II on brain monocytes. Additionally, we found an increase in Ly-6Chigh monocyte frequency and decreased CD44 expression on CD8+ and CD4+ T cells in PGRN KO female blood. Given that Gpnmb, which encodes for the lysosomal protein Glycoprotein non-metastatic melanoma protein B, has been reported to be upregulated in PGRN KO mice, we investigated changes in GPNMB protein expression associated with PGRN deficits and found that GPNMB is modulated in myeloid cells in a sex-specific manner. Discussion: Our data suggest that PGRN and GPNMB jointly regulate the peripheral and the central immune system in a sex-specific manner; thus, understanding their associated mechanisms could pave the way for developing new neuroprotective strategies to modulate central and peripheral inflammation to lower risk for neurodegenerative diseases and possibly delay or halt progression.

Experimental colitis promotes sustained, sex-dependent, T-cell-associated neuroinflammation and parkinsonian neuropathology.

BACKGROUND: The etiology of sporadic Parkinson's disease (PD) remains uncertain, but genetic, epidemiological, and physiological overlap between PD and inflammatory bowel disease suggests that gut inflammation could promote dysfunction of dopamine-producing neurons in the brain. Mechanisms behind this pathological gut-brain effect and their interactions with sex and with environmental factors are not well understood but may represent targets for therapeutic intervention. METHODS: We sought to identify active inflammatory mechanisms which could potentially contribute to neuroinflammation and neurological disease in colon biopsies and peripheral blood immune cells from PD patients. Then, in mouse models, we assessed whether dextran sodium sulfate-mediated colitis could exert lingering effects on dopaminergic pathways in the brain and whether colitis increased vulnerability to a subsequent exposure to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We assessed the involvement of inflammatory mechanisms identified in the PD patients in colitis-related neurological dysfunction in male and female mice, utilizing mice lacking the Regulator of G-Protein Signaling 10 (RGS10)-an inhibitor of nuclear factor kappa B (NFκB)-to model enhanced NFκB activity, and mice in which CD8+ T-cells were depleted. RESULTS: High levels of inflammatory markers including CD8B and NFκB p65 were found in colon biopsies from PD patients, and reduced levels of RGS10 were found in immune cells in the blood. Male mice that experienced colitis exhibited sustained reductions in tyrosine hydroxylase but not in dopamine as well as sustained CD8+ T-cell infiltration and elevated Ifng expression in the brain. CD8+ T-cell depletion prevented colitis-associated reductions in dopaminergic markers in males. In both sexes, colitis potentiated the effects of MPTP. RGS10 deficiency increased baseline intestinal inflammation, colitis severity, and neuropathology. CONCLUSIONS: This study identifies peripheral inflammatory mechanisms in PD patients and explores their potential to impact central dopaminergic pathways in mice. Our findings implicate a sex-specific interaction between gastrointestinal inflammation and neurologic vulnerability that could contribute to PD pathogenesis, and they establish the importance of CD8+ T-cells in this process in male mice.

Protein Composition and Associated Material Properties of Cobweb Spiders' Gumfoot Glue Droplets.

The origin of aggregate silk glands and their production of wet adhesive silks is considered a key innovation of the Araneoidea, a superfamily of spiders that build orb-webs and cobwebs. Orb-web weavers place aggregate glue on an extensible capture spiral, whereas cobweb weavers add it to the ends of strong, stiff fibers, called gumfoot lines. Here we describe the material behavior and quantitative proteomics of the aggregate glues of two cobweb weaving species, the western black widow, Latrodectus hesperus, and the common house spider, Parasteatoda tepidariorum. For each species, respectively, we identified 48 and 33 proteins that were significantly more abundant in the portion of the gumfoot line with glue than in its fibers. These proteins were more highly glycosylated and phosphorylated than proteins found in silk fibers without glue, which likely explains aggregate glue stickiness. Most glue-enriched proteins were of anterior aggregate gland origin, supporting the hypothesis that cobweb weavers' posterior aggregate glue is specialized for another function. We found that cobweb weaver glue droplets are stiffer and tougher than the adhesive of most orb-web weaving species. Attributes of gumfoot glue protein composition that likely contribute to this stiffness include the presence of multiple protein families with conserved cysteine residues, a bimodal distribution of isoelectric points, and families with conserved functions in protein aggregation, all of which should contribute to cohesive protein-protein interactions. House spider aggregate droplets were more responsive to humidity changes than black widow droplets, which could be mediated by differences in protein sequence, post-translational modifications, the non-protein components of the glue droplets, and/or the larger amount of aqueous material that surrounds the adhesive cores of their glue droplets.

Adolescent stress sensitizes the adult neuroimmune transcriptome and leads to sex-specific microglial and behavioral phenotypes.

Adolescent exposure to chronic stress, a risk factor for mood disorders in adulthood, sensitizes the neuroinflammatory response to a subsequent immune challenge. We previously showed that chronic adolescent stress (CAS) in rats led to distinct patterns of neuroimmune priming in adult male and female rats. However, sex differences in the neuroimmune consequences of CAS and their underlying mechanisms are not fully understood. Here we hypothesized that biological sex would dictate differential induction of inflammation-related transcriptomic pathways and immune cell involvement (microglia activation and leukocyte presence) in the hippocampus of male and female rats with a history of CAS. Adolescent rats underwent CAS (six restraint and six social defeat episodes during postnatal days 38-49), and behavioral assessments were conducted in adolescence and adulthood. Neuroimmune measures were obtained following vehicle or a systemic lipopolysaccharide (LPS) challenge in adulthood. CAS led to increased time in the corners of the open field in adolescence. In males, CAS also increased social avoidance. As adults, CAS rats displayed an exaggerated enrichment of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway and chemokine induction following LPS challenge, and increased number of perivascular CD45+ cells in the hippocampus. However, CAS females, but not males, showed exaggerated glucocorticoid receptor (GR) pathway enrichment and increased microglial complexity. These results provide further insight to the mechanisms by which peripheral immune events may influence neuroimmune responses differentially among males and females and further demonstrate the importance of adolescent stress in shaping adult responses.

Transgenic Mice Expressing Human α-Synuclein in Noradrenergic Neurons Develop Locus Ceruleus Pathology and Nonmotor Features of Parkinson's Disease.

Degeneration of locus ceruleus (LC) neurons and dysregulation of noradrenergic signaling are ubiquitous features of Parkinson's disease (PD). The LC is among the first brain regions affected by α-synuclein (asyn) pathology, yet how asyn affects these neurons remains unclear. LC-derived norepinephrine (NE) can stimulate neuroprotective mechanisms and modulate immune cells, while dysregulation of NE neurotransmission may exacerbate disease progression, particularly nonmotor symptoms, and contribute to the chronic neuroinflammation associated with PD pathology. Although transgenic mice overexpressing asyn have previously been developed, transgene expression is usually driven by pan-neuronal promoters and thus has not been selectively targeted to LC neurons. Here we report a novel transgenic mouse expressing human wild-type asyn under control of the noradrenergic-specific dopamine ß-hydroxylase promoter (DBH-hSNCA). These mice developed oligomeric and conformation-specific asyn in LC neurons, alterations in hippocampal and LC microglial abundance, upregulated GFAP expression, degeneration of LC fibers, decreased striatal DA metabolism, and age-dependent behaviors reminiscent of nonmotor symptoms of PD that were rescued by adrenergic receptor antagonists. These mice provide novel insights into how asyn pathology affects LC neurons and how central noradrenergic dysfunction may contribute to early PD pathophysiology.SIGNIFICANCE STATEMENT ɑ-Synuclein (asyn) pathology and loss of neurons in the locus ceruleus (LC) are two of the most ubiquitous neuropathologic features of Parkinson's disease (PD). Dysregulated norepinephrine (NE) neurotransmission is associated with the nonmotor symptoms of PD, including sleep disturbances, emotional changes such as anxiety and depression, and cognitive decline. Importantly, the loss of central NE may contribute to the chronic inflammation in, and progression of, PD. We have generated a novel transgenic mouse expressing human asyn in LC neurons to investigate how increased asyn expression affects the function of the central noradrenergic transmission and associated behaviors. We report cytotoxic effects of oligomeric and conformation-specific asyn, astrogliosis, LC fiber degeneration, disruptions in striatal dopamine metabolism, and age-dependent alterations in nonmotor behaviors without inclusions.

Chronic psychological stress during adolescence induces sex-dependent adulthood inflammation, increased adiposity, and abnormal behaviors that are ameliorated by selective inhibition of soluble tumor necrosis factor with XPro1595.

Physical and psychosocial maltreatment experienced before the age of 18, termed early life adversity (ELA), affects an estimated 39% of the world's population, and has long-term detrimental health and psychological outcomes. While adult phenotypes vary following ELA, inflammation and altered stress responsivity are pervasive. Cytokines, most notably tumor necrosis factor (TNF), are elevated in adults with a history of ELA. While soluble TNF (solTNF) drives chronic inflammatory disease, transmembrane TNF facilitates innate immunity. Here, we test whether solTNF mediates the behavioral and molecular outcomes of adolescent psychological stress by administering a brain permeable, selective inhibitor of solTNF, XPro1595. Male and female C57BL/6 mice were exposed to an aggressive rat through a perforated translucent ball ('predatory stress') or transported to an empty room for 30 min for 30 days starting on postnatal day 34. Mice were given XPro1595 or vehicle treatment across the last 15 days. Social interaction, sucrose preference, and plasma inflammation were measured at 2 and 4 weeks, and open field behavior, adiposity, and neuroinflammation were measured at 4 weeks. Chronic adolescent stress resulted in increased peripheral inflammation and dysregulated neuroinflammation in adulthood in a sex-specific manner. Abnormal social and open field behavior, fat pad weight, and fecal boli deposition were noted after 30 days; solTNF antagonism ameliorated the effects of stress. Together, these data support our hypothesis, and suggest that targeting solTNF with XPro1595 may improve quality of life for individuals with a history of adolescent stress.

Orb weaver glycoprotein is a smart biological material, capable of repeated adhesion cycles.

Orb weavers produce webs that trap prey using a capture spiral formed of regularly spaced glue droplets supported by protein fibers. Each droplet consists of an outer aqueous layer and an adhesive, viscoelastic glycoprotein core. Organic and inorganic compounds in the aqueous layer make droplets hygroscopic and cause droplet features to change with environmental humidity. When droplets contact a surface, they adhere and extend as an insect struggles. Thus, a droplet's extensibility is as important for prey capture as its adhesion. Cursory observations show that droplets can adhere, extend, and pull off from a surface several times, a process called cycling. Our study cycled individual droplets of four species-Argiope aurantia, Neoscona crucifera, Verrucosa arenata, and Larinioides cornutus. Droplets were subjected to 40 cycles at two humidities to determine how humidity affected droplet performance. We hypothesized that droplets would continue to perform, but that performance would decrease. Droplet performance was characterized by filament length and force on droplets at pull-off, aqueous volume, and glycoprotein volume. As hypothesized, cycling decreased performance, notably extensibility and aqueous volume. However, humidity did not impact the response to cycling. In a natural context, droplets are not subjected to extensive cycling, but reusability is advantageous for orb-weaving spiders. Moreover, the ability to cycle, combined with their environmental responsiveness, allows us to characterize orb weaver droplets as smart materials for the first time.

Chronic adolescent stress sex-specifically alters the hippocampal transcriptome in adulthood.

Chronic adolescent stress alters behavior in a sex-specific manner at the end of adolescence and in adulthood. Although prolonged behavioral repercussions of chronic adolescent stress have been documented, the potential underlying mechanisms are incompletely understood. In this study we demonstrate that a history of chronic adolescent stress modified the adult stress response, as measured by corticosterone concentration, such that a history of chronic adolescent stress resulted in a blunted response to a novel acute stressor. In order to begin to address potential mechanistic underpinnings, we assessed the extent to which chronic adolescent stress impacted global DNA methylation. Reduced global hippocampal methylation was evident in females with a history of chronic adolescent stress; thus, it was possible that chronic adolescent stress altered global transcription in the whole hippocampi of adult male and female rats. In addition, because acute stress can stimulate a genomic response, we assessed the transcriptome following exposure to an acute novel stressor to determine the extent to which a history of chronic adolescent stress modifies the adult transcriptional response to an acute stressor in males and females. In addition to the reduction in global methylation, chronic adolescent stress resulted in distinct patterns of gene expression in the adult hippocampus that differentiated by sex. Furthermore, both sex and a history of chronic adolescent stress influenced the transcriptional response to an acute novel stressor in adulthood, suggesting both latent and functional effects of chronic adolescent stress at the level of gene transcription. Pathway analysis indicated that ESR1 and IFN-α may be particularly influential transcription factors mediating these transcriptional differences and suggest candidate mechanisms for future studies. Collectively, these studies demonstrate sex-specific and enduring effects of adolescent stress exposure that are more pronounced in females than in males.

Chronic adolescent stress sex-specifically alters central and peripheral neuro-immune reactivity in rats.

Adversity during development is a reliable predictor of psychiatric disorders such as depression and anxiety which are increasingly recognized to have an immune component. We have previously demonstrated that chronic adolescent stress (CAS) in rats leads to depressive-like behavior in adulthood along with long-lasting changes to the hypothalamic-pituitary-adrenal axis and pro-inflammatory cytokine induction in the hippocampus. However, the mechanisms by which CAS promotes hippocampal inflammation are not yet defined. Here we tested the hypothesis that a history of CAS exaggerates induction of the pro-inflammatory NFκB pathway in the adult rat hippocampus without compromising the peripheral immune response. We also assessed potential sex differences because it is unclear whether females, who are twice as likely to suffer from mood disorders as males, are disproportionally affected by stress-primed inflammation. Male and female adolescent rats underwent a CAS paradigm or received no stress. Six weeks following the last stressor, all rats received a single systemic injection of either lipopolysaccharide or vehicle to unmask possible immune-priming effects of CAS. An NFκB signaling PCR array demonstrated that CAS exaggerated the expression of NFκB-related genes in the hippocampus of both males and females. Interestingly, targeted qPCR demonstrated that CAS potentiated the induction of hippocampal IL1B and REL mRNA in female rats only, suggesting that some immune effects of CAS are indeed sex-specific. In contrast to the hippocampal findings, indices of peripheral inflammation such as NFκB activity in the spleen, plasma IL-1ß, IL-6, TNF-α, and corticosterone were not impacted by CAS in female rats. Despite showing no pro-inflammatory changes to hippocampal mRNA, male CAS rats displayed lower plasma corticosterone response to LPS at 2 h after injection followed by an exaggerated plasma IL-1ß response at 4 h. This potentially blunted corticosterone response coupled with excessive innate immune signaling in the periphery is consistent with possible glucocorticoid resistance in males. In contrast, the effects of CAS manifested as excessive hippocampal immune reactivity in females. We conclude that while a history of exposure to chronic adolescent stress enhances adult immune reactivity in both males and females, the mechanism and manifestation of such alterations are sex-specific.

HIV and symptoms of depression are independently associated with impaired glucocorticoid signaling.

Chronic inflammation caused by HIV infection may lead to deficient glucocorticoid (GC) signaling predisposing people living with HIV to depression and other psychiatric disorders linked to GC resistance. We hypothesized that comorbid HIV and depressive symptoms in women would synergistically associate with deficits in GC signaling. This cross-sectional study used samples obtained from the Women's Interagency HIV Study (WIHS). The Centers for Epidemiological Studies (CES-D) was used to define depression in four groups of women from the Women's Interagency HIV Study (WIHS): 1) HIV-negative, non-depressed (n = 37); 2) HIV-negative, depressed (n = 34); 3) HIV-positive, non-depressed (n = 38); and 4) HIV-positive, depressed (n = 38). To assess changes in GC signaling from peripheral blood mononuclear cells (PBMCs), we examined baseline and dexamethasone (Dex)-stimulated changes in the expression of the GC receptor (GR, gene: Nr3c1) and its negative regulator Fkbp5 via quantitative RT-PCR. GR sensitivity was evaluated in vitro by assessing the Dex inhibition of lipopolysaccharide (LPS)-stimulated IL-6 and TNF-α levels. Depressive symptoms and HIV serostatus were independently associated with elevated baseline expression of Fkbp5 and Nr3c1. Depressive symptoms, but not HIV status, was independently associated with reduced LPS-induced release of IL-6. Counter to predictions, there was no interactive association of depressive symptoms and HIV on any outcome. Comorbid depressive symptoms with HIV infection were associated with a gene expression and cytokine profile similar to that of healthy control women, a finding that may indicate further disruptions in disease adaptation.

Measuring corticosterone concentrations over a physiological dynamic range in female rats.

Accurate assessment of plasma corticosterone, the primary stress hormone in rodents, is an essential part of characterizing the stress response in experimental animals. To this end, both enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) remain widely used. However, considerable assay-specific variability exists among commercially available corticosterone assays due to differing assay principles, detection methods, range, and sensitivity. While technical comparisons of commercially available corticosterone assays have previously been conducted, the ability to detect acute stress-induced endocrine changes has not been compared among these methods to date. Using the forced swim test, a commonly utilized behavioral paradigm in rodents as a physiologically-relevant acute stress challenge, we compared four commercial corticosterone assays - three ELISA kits and one RIA kit - in their ability to detect corticosterone across a dynamic range of both baseline and acute swim stress-driven concentrations. While all methods yielded results that were consistent at measuring relative differences between samples, only two of the four assays evaluated detected a statistically significant increase in corticosterone in rats exposed to acute swim stress compared to rats at baseline. The ELISA kit from Enzo Life Sciences demonstrated the greatest percent increase in plasma corticosterone from baseline to acute stress conditions. The RIA kit from MP Biomedicals also detected a significant corticosterone increase and yielded higher concentrations of corticosterone both at baseline and in the acute stress condition relative to the other three assays. We conclude that choice of assay can impact interpretation of data due to differences in efficacy across a dynamic range of physiological concentrations of corticosterone.

Prenatal stress-induced increases in hippocampal von Willebrand factor expression are prevented by concurrent prenatal escitalopram.

Prenatal stress has been linked to deficits in neurological function including deficient social behavior, alterations in learning and memory, impaired stress regulation, and susceptibility to adult disease. In addition, prenatal environment is known to alter cardiovascular health; however, limited information is available regarding the cerebrovascular consequences of prenatal stress exposure. Vascular disturbances late in life may lead to cerebral hypoperfusion which is linked to a variety of neurodegenerative and psychiatric diseases. The known impact of cerebrovascular compromise on neuronal function and behavior highlights the importance of characterizing the impact of stress on not just neurons and glia, but also cerebrovasculature. Von Willebrand factor has previously been shown to be impacted by prenatal stress and is predictive of cerebrovascular health. Here we assess the impact of prenatal stress on von Willebrand factor and related angiogenic factors. Furthermore, we assess the potential protective effects of concurrent anti-depressant treatment during in utero stress exposure on the assessed cerebrovascular endpoints. Prenatal stress augmented expression of von Willebrand factor which was prevented by concurrent in utero escitalopram treatment. The functional implications of this increase in von Willebrand factor remain elusive, but the presented data demonstrate that although prenatal stress did not independently impact total vascularization, exposure to chronic stress in adulthood decreased blood vessel length. In addition, the current study demonstrates that production of reactive oxygen species in the hippocampus is decreased by prenatal exposure to escitalopram. Collectively, these findings demonstrate that the prenatal experience can cause complex changes in adult cerebral vascular structure and function.

Reduced marker of vascularization in the anterior hippocampus in a female monkey model of depression.

Depression is a common and debilitating mood disorder that impacts women more often than men. The mechanisms that result in depressive behaviors are not fully understood; however, the hippocampus has been noted as a key structure in the pathophysiology of depression. In addition to neural implications of depression, the cardiovascular system is impacted. Although not as commonly considered, the cerebrovasculature is critical to brain function, impacted by environmental stimuli, and is capable of altering neural function and thereby behavior. In the current study, we assessed the relationship between depressive behavior and a marker of vascularization of the hippocampus in adult female cynomolgus macaques (Macaca fascicularis). Similar to previously noted impacts on neuropil and glia, the depressed phenotype predicts a reduction in a marker of vascular length in the anterior hippocampus. These data reinforce the growing recognition of the effects of depression on vasculature and support further consideration of vascular endpoints in studies aimed at the elucidation of the mechanisms underlying depression.

Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress.

Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was increased in the hippocampus. In addition, adolescent stress exposure increased microglial branching and junctions in female wild-type rats without causing any additional increase in HIV-1 rats. These data suggest that the presence of HIV-1 proteins during development leads to alterations in behavioral and neuroinflammatory endpoints that are not further impacted by concurrent chronic adolescent stress.

Brief anesthesia by isoflurane alters plasma corticosterone levels distinctly in male and female rats: Implications for tissue collection methods.

Euthanasia by anesthetic agents is commonly performed prior to tissue collection in order to minimize pain and distress to the animal. However, depending on their mechanism of action as well as administration regimen, different methods of anesthesia may trigger an acute stress response through engaging the hypothalamic-pituitary-adrenal (HPA) axis, which can impact numerous other physiological processes that the researcher may wish to examine as endpoints. We investigated the effects of the commonly used anesthetic agent isoflurane on two different endpoints related to the stress response: plasma corticosterone levels and gene expression of the glucocorticoid receptor (GR) as well as several of its regulators including FK506-binding protein 51 (Fkbp5) in the hippocampus of male and female rats. Our results indicate that brief exposure to anesthesia by isoflurane prior to decapitation can alter plasma corticosterone levels differentially in male and female rats within minutes without impacting gene expression in the hippocampus. We conclude that collection methods can influence stress-related physiological endpoints in female rats and the potential influence of even brief anesthesia as well as sex differences in response to anesthesia should be evaluated during the experimental design process and data interpretation. This finding is particularly important in light of new NIH standards regarding sex and reproducibility, and care should be taken to be certain that sex differences in endpoints of interest are not an artifact of sex differences in response to collection paradigms.