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Background & Aims Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis. Methods Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages. Results We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process. Conclusions In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction., Graphical abstract

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression)., Highlights • Microglia can be preferentially shaken off from primary mixed and secondary cultures. • Secondary cultures contaminated mainly by astrocytes (12–19%) and neurons (2–3%). • Tertiary cultures had low cell yields (x̄ = 5.1% for T1, x̄ = 2.4% for T2). • Tertiary cultures had consistently high purity (x̄ = 93.1%; 99% in 1 of 7 attempts). • Tertiary cultures are suitable for gene or protein expression analyses.

The assembly of tau protein into abnormal filaments and brain cell degeneration are characteristic of a number of human neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Several murine models have been generated to better understand the mechanisms contributing to tau assembly and neurodegeneration. Taking advantage of the more elaborate central nervous system and higher cognitive abilities of the rat, we generated a model expressing the longest human tau isoform (2N4R) with the P301S mutation. This transgenic rat line, R962-hTau, exhibits the main features of human tauopathies, such as: age-dependent increase in inclusions comprised of aggregated-tau, neuronal loss, global neurodegeneration as reflected by brain atrophy and ventricular dilation, alterations in astrocytic and microglial morphology, and myelin loss. In addition, substantial deficits across multiple memory and learning paradigms, including novel object recognition, fear conditioning and Morris water maze tasks, were observed at the time of advanced tauopathy. These results support the concept that progressive tauopathy correlates with brain atrophy and cognitive impairment., Highlights • A transgenic rat model expressing the full length human tau with the P301S mutation. • Progressive human-like tau brain pathology with recruitment of endogenous rodent tau. • Pathology-induced gliosis, myelin breakdown, neuronal loss and ventricular dilation. • Widespread behavioural deficits occur at end-stage tau pathology.

Highlights • This study is the first to examine HAP1-expression in dorsal root ganglia (DRG). • HAP1 is highly co-expressed with the markers of nociceptive/proprioceptive neurons. • HAP1 is completely lacking in the touch-sensitive DRG neurons. • HAP1 may play an important role in modulating nociceptive/proprioceptive functions. • It will be of great interest to clarify the pathophysiological role of HAP1 in DRG., Huntingtin-associated protein 1 (HAP1) is a polyglutamine (polyQ) length-dependent interactor with causal agents in several neurodegenerative diseases and has been regarded as a protective factor against neurodegeneration. In normal rodent brain and spinal cord, HAP1 is abundantly expressed in the areas that are spared from neurodegeneration while those areas with little HAP1 are frequent targets of neurodegeneration. We have recently showed that HAP1 is highly expressed in the spinal dorsal horn and may participate in modification/protection of certain sensory functions. Neurons in the dorsal root ganglia (DRG) transmits sensory stimuli from periphery to spinal cord/brain stem. Nevertheless, to date HAP1 expression in DRG remains unreported. In this study, the expression of HAP1 in cervical, thoracic, lumbar and sacral DRG in adult male mice and its relationships with different chemical markers for sensory neurons were examined using Western blot and immunohistochemistry. HAP1-immunoreactivity was detected in the cytoplasm of DRG neurons, and the percentage of HAP1-immunoreactive (ir) DRG neurons was ranged between 28–31 %. HAP1-immunoreactivity was comparatively more in the small cells (47–58 %) and medium cells (40–44 %) than that in the large cells (9–11 %). Double-immunostaining for HAP1 and markers for nociceptive or mechanoreceptive neurons showed that about 70–80 % of CGRP-, SP-, CB-, NOS-, TRPV1-, CR- and PV-ir neurons expressed HAP1. In contrast, HAP1 was completely lacking in TH-ir neurons. Our current study is the first to clarify that HAP1 is highly expressed in nociceptive/proprioceptive neurons but absent in light-touch-sensitive TH neurons, suggesting the potential importance of HAP1 in pain transduction and proprioception.

Microglia are the predominant immune cells of the central nervous system (CNS) that exert key physiological roles required for maintaining CNS homeostasis, notably in response to chronic stress, as well as mediating synaptic plasticity, learning and memory. The repeated exposure to stress confers a higher risk of developing neurodegenerative diseases including sporadic Alzheimer's disease (AD). While microglia have been causally linked to amyloid beta (Aβ) accumulation, tau pathology, neurodegeneration, and synaptic loss in AD, they were also attributed beneficial roles, notably in the phagocytic elimination of Aβ. In this review, we discuss the interactions between chronic stress and AD pathology, overview the roles played by microglia in AD, especially focusing on chronic stress as an environmental risk factor modulating their function, and present recently-described microglial phenotypes associated with neuroprotection in AD. These microglial phenotypes observed under both chronic stress and AD pathology may provide novel opportunities for the development of better-targeted therapeutic interventions. Keywords: Microglia, Stress, Alzheimer's disease, Neurodegeneration, Neuroinflammation, Synaptic remodeling, Microglial phenotypes, Dark microglia

Highlights • We identified XCR1 in the peripheral and central nervous systems and demonstrated its upregulation following nerve injury. • In injured nerve, XCR1 is present in nerve fibers, CD45-positive leucocytes and Schwann cells. • In Vc, XCR1 labeling is consistent with expression in terminals of Aδ- and C-fiber afferents and excitatory interneurons. • XCL1 increases neuronal excitability and activates intracellular signaling in Vc, a pain-processing region of the CNS. • These data provide the first evidence that the XCL1-XCR1 axis may play a role in trigeminal pain pathways., Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

Introduction There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid β (Aβ)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD. Methods The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aβ burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze. Results Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aβ deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice. Conclusion In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs., Highlights • Methysticin activates the Nrf2/ARE system in the hippocampus of mice. • Methysticin protects AD mice against oxidative stress and associated neuroinflammation due to Nrf2 activation. • Methysticin improves long-term memory impairment in this mouse model of AD.

Highlights • Seasonal changes in rat trigger change in hypothalamic histone deacetylases (HDACs). • NF-κB is an inflammatory regulator under seasonal control in the hypothalamus. • These HDACs may control hypothalamic inflammatory and nuclear receptor pathways., Seasonal animals undergo changes in physiology and behavior between summer and winter conditions. These changes are in part driven by a switch in a series of hypothalamic genes under transcriptional control by hormones and, of recent interest, inflammatory factors. Crucial to the control of transcription are histone deacetylases (HDACs), generally acting to repress transcription by local histone modification. Seasonal changes in hypothalamic HDAC transcripts were investigated in photoperiod-sensitive F344 rats by altering the day-length (photoperiod). HDAC4, 6 and 9 were found to change in expression. The potential influence of HDACs on two hypothalamic signaling pathways that regulate transcription, inflammatory and nuclear receptor signaling, was investigated. For inflammatory signaling the focus was on NF-κB because of the novel finding made that its expression is seasonally regulated in the rat hypothalamus. For nuclear receptor signaling it was discovered that expression of retinoic acid receptor beta was regulated seasonally. HDAC modulation of NF-κB-induced pathways was examined in a hypothalamic neuronal cell line and primary hypothalamic tanycytes. HDAC4/5/6 inhibition altered the control of gene expression (Fos, Prkca, Prkcd and Ptp1b) by inducers of NF-κB that activate inflammation. These inhibitors also modified the action of nuclear receptor ligands thyroid hormone and retinoic acid. Thus seasonal changes in HDAC4 and 6 have the potential to epigenetically modify multiple gene regulatory pathways in the hypothalamus that could act to limit inflammatory pathways in the hypothalamus during long-day summer-like conditions.

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid-Schiff-diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD.

Objective Obesity has been linked to an inflammation like state in the hypothalamus, mainly characterized by reactive gliosis (RG) of astrocytes and microglia. Here, using two diet models or pharmacological treatment, we assessed the effects of mild and drastic weight loss on RG, in the context of high-fat diet (HFD) induced obesity. Methods We subjected HFD-induced obese (DIO) male C57BL/6J mice to a weight loss intervention with a switch to standard chow, calorie restriction (CR), or treatment with the Glp1 receptor agonist Exendin-4 (EX4). The severity of RG was estimated by an ordinal scoring system based on fluorescence intensities of glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1 positive (Iba1), cell numbers, and morphological characteristics. Results In contrast to previous reports, DIO mice fed chronically with HFD showed no differences in microglial or astrocytic RG, compared to chow controls. Moreover, mild or profound weight loss had no impact on microglial RG. However, astrocyte RG was increased in CR and EX4 groups compared to chow fed animals and strongly correlated to body weight loss. Profound weight loss by either CR or EX4 was further linked to increased levels of circulating non-esterified free fatty acids. Conclusions Overall, our data demonstrate that in a chronically obese state, astrocyte and microglial RG is indifferent from that observed in age-matched chow controls. Nonetheless, profound acute weight loss can induce astrocyte RG in the hypothalamic arcuate nucleus, possibly due to increased circulating NEFAs. This suggests that astrocytes may sense acute changes to both the dietary environment and body weight., Graphical abstract Image 1, Highlights • Acute weight loss induces reactive gliosis in arcuate nucleus residing astrocytes. • Reactive gliosis after chronic high-fat diet is comparable to chow fed litter mates. • Observed gliosis coincides with increased circulating non-esterified fatty acids.