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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.

Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, most studies have examined the role of glia activation within the spinal cord. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This review attempts to summarize what is known regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance. These data, together, provide information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain.

Stroke is a multiphasic process involving a direct ischemic brain injury which is then exacerbated by the influx of immune cells into the brain tissue. Activation of brain endothelial cells leads to the expression of adhesion molecules such vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells, further increasing leukocyte recruitment. Polymerase δ-interacting protein 2 (Poldip2) promotes brain vascular inflammation and leukocyte recruitment via unknown mechanisms. This study aimed to define the role of Poldip2 in mediating vascular inflammation and leukocyte recruitment following cerebral ischemia. Cerebral ischemia was induced in Poldip2+/+ and Poldip2+/− mice and brains were isolated and processed for flow cytometry or RT-PCR. Cultured rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion on focal adhesion kinase (FAK)-mediated VCAM-1 induction. Poldip2 depletion in vivo attenuated the infiltration of myeloid cells, inflammatory monocytes/macrophages and decreased the induction of adhesion molecules. Focusing on VCAM-1, we demonstrated mechanistically that FAK activation was a critical intermediary in Poldip2-mediated VCAM-1 induction. In conclusion, Poldip2 is an important mediator of endothelial dysfunction and leukocyte recruitment. Thus, Poldip2 could be a therapeutic target to improve morbidity following ischemic stroke.

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 β-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.

Opioid tolerance and the potential for addiction is a significant burden associated with pain management, yet its precise underlying mechanism and prevention remain elusive. Immune signaling contributes to the decreased efficacy of opioids, and we recently demonstrated that Toll-like receptor 4 (TLR4)-mediated neuroinflammation in the periaqueductal gray (PAG) drives tolerance. Tumor necrosis factor (TNF), a product of TLR4 signaling, promotes inflammation and facilitates glutamatergic signaling, key components of opioid tolerance. Therefore, we hypothesize that TLR4-mediated opioid tolerance requires TNF signaling. By expression of a dominant-negative TNF peptide via lentiviral vector injection in rat PAG to sequester soluble TNF (solTNF), we demonstrate that solTNF mediates morphine tolerance induced by TLR4 signaling, stimulates neuroinflammation (increased IL-1β and TLR4 mRNA), and disrupts glutamate reuptake (decreased GLT-1 and GLAST mRNA). We further demonstrate the efficacy of the brain-permeant PEGylated version of the anti-solTNF peptide, XPro1595, injected systemically, to normalize morphine-induced CNS neuroinflammation and morphine- and endotoxin-induced changes in glutamate transport, effectively preserving the efficacy of morphine analgesia and eliminating tolerance. Our findings provide a novel pharmacological target for the prevention of opioid-induced immune signaling, tolerance, and addiction.

Morphine remains one of the most widely prescribed opioids for alleviation of persistent and/or severe pain; however, multiple preclinical and clinical studies report that morphine is less efficacious in females compared to males. Morphine primarily binds to the mu opioid receptor, a prototypical G-protein coupled receptor densely localized in the midbrain periaqueductal gray. Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray, and its descending projections to the rostral ventromedial medulla and spinal cord, as an essential descending inhibitory circuit mediating opioid-based analgesia. Remarkably, the majority of studies published over the following 30 years were conducted in males with the implicit assumption that the anatomical and physiological characteristics of this descending inhibitory circuit were comparable in females; not surprisingly, this is not the case. Several factors have since been identified as contributing to the dimorphic effects of opioids, including sex differences in the neuroanatomical and neurophysiological characteristics of the descending inhibitory circuit and its modulation by gonadal steroids. Recent data also implicate sex differences in opioid metabolism and neuroimmune signaling as additional contributing factors. Here we cohesively present these lines of evidence demonstrating a neural basis for sex differences in opioid modulation of pain, with a focus on the PAG as a sexually dimorphic core of descending opioid-induced inhibition and argue for the development of sex-specific pain therapeutics.

Recent studies about the gut-brain axis suggest that high fat high fructose (HFHF) consumption is associated with chronic peripheral inflammation due to disruption of gut flora. Some evidence sugge...

The ventrolateral periaqueductal gray (vlPAG) is an integral locus for morphine action. Although it is clear that glia contribute to the development of morphine tolerance, to date, the investigation of their role has been limited to spinal and medullary loci. Opioids induce a neuroinflammatory response that opposes acute and long-term analgesia, thereby limiting their efficacy as therapeutic agents. Recent data suggest that the innate immune receptor Toll-like receptor 4 (TLR4), along with its coreceptor myeloid differentiation factor-2 (MD-2), mediates these effects. To date, the brain loci through which TLR4 modulates morphine tolerance have not been identified. We have previously demonstrated that chronic subcutaneous morphine results in tolerance that is accompanied by increases in vlPAG glial cell activity. Usingin vivopharmacological manipulations of vlPAG glia and TLR4 in the adult male rat, we show that intra-vlPAG administration of the general glial cell metabolic inhibitor propentofylline or the astrocyte activity inhibitor fluorocitrate attenuate tolerance to morphine. Characterization of MD-2 expression within the PAG revealed dense MD-2 expression throughout the vlPAG. Further, antagonizing vlPAG TLR4 dose dependently prevented the development of morphine tolerance, and vlPAG microinjections of TLR4 agonists dose dependently produced a “naive” tolerance to subsequent challenge doses of morphine. Finally, using a model of persistent inflammatory pain and pharmacological manipulation of TLR4 we demonstrate that systemic antagonism of TLR4 potentiated acute morphine antihyperalgesia. These results, together, indicate that vlPAG glia regulate morphine tolerance development via TLR4 signaling, and implicate TLR4 as a potential therapeutic target for the treatment of pain.

Morphine is among the most prevalent analgesics prescribed for chronic pain. However, prolonged morphine treatment results in the development of analgesic tolerance. An abundance of evidence has accumulated indicating that central nervous system glial cell activity facilitates pain transmission and opposes morphine analgesia. While the midbrain ventrolateral periaqueductal gray (vlPAG) is an important neural substrate mediating pain modulation and the development of morphine tolerance, no studies have directly assessed the role of PAG glia. Here we test the hypothesis that morphine-induced increases in vlPAG glial cell activity contribute to the development of morphine tolerance. As morphine is primarily consumed for the alleviation of severe pain, the influence of persistent inflammatory pain was also assessed. Administration of morphine, in the absence of persistent inflammatory pain, resulted in the rapid development of morphine tolerance and was accompanied by a significant increase in vlPAG glial activation. In contrast, persistent inflammatory hyperalgesia, induced by intraplantar administration of complete Freund’s adjuvant (CFA), significantly attenuated the development of morphine tolerance. No significant differences were noted in vlPAG glial cell activation for CFA-treated animals versus controls. These results indicate that vlPAG glia are modulated by a persistent pain state, and implicate vlPAG glial cells as possible regulators of morphine tolerance. Perspective The development of morphine tolerance represents a significant impediment to its use in the management of chronic pain. We report that morphine tolerance is accompanied by increased glial cell activation within the vlPAG, and that the presence of a persistent pain state prevented vlPAG glial activation and attenuated morphine tolerance.

Proceptive and receptive behaviors of female rodents, such as golden hamsters, are often regulated by changes in circulating levels of ovarian hormones. However, less is known about how ovarian hormones might regulate female hamster's attraction and preference for volatile odor from males. To evaluate this, we assessed female preference by recording investigation and proximity to male and female volatile odorants in a Y-maze across all days of the estrous cycle (Experiments 1 and 2) or following ovariectomy (Experiment 3). In Experiment 1, female subjects were tested four times, once on each day of their estrous cycle. Females showed a preference for male odors on diestrus day 1 and to a lesser degree on proestrus, but showed no preference on the day of behavioral estrus. Irrespective of cycle day, preference was apparent in the first few days of testing and disappeared by the fourth day, suggesting that repeated testing attenuated female preference. To avoid this problem, in Experiment 2 each animal was tested only on one day of the 4-day estrous cycle. Female preference for male volatile odors over those from females was observed on each day of their estrous cycle, including estrus. Moreover, following gonadectomy (Experiment 3) female hamsters still preferred male volatile odors to those of females. Taken together, this suggests that circulating levels of gonadal hormones do not influence preference for male volatile odors in female hamsters.