Probes for INS

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR+ neurons and promote chronic itch.RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR+ neuron interactions.We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR+ neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β+ microglia are localized in close proximity to GRPR+ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR+ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR+ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.

Nonconvulsive epileptiform activity and microglial alterations have been detected in people with Alzheimer’s disease (AD) and related mouse models. However, the relationship between these abnormalities remains to be elucidated. We suppressed epileptiform activity by treatment with the antiepileptic drug levetiracetam or by genetic ablation of tau and found that these interventions reversed or prevented aberrant microglial gene expression in brain tissues of aged human amyloid precursor protein transgenic mice, which simulate several key aspects of AD. The most robustly modulated genes included multiple factors previously implicated in AD pathogenesis, including TREM2, the hypofunction of which increases disease risk. Genetic reduction of TREM2 exacerbated epileptiform activity after mice were injected with kainate. We conclude that AD-related epileptiform activity markedly changes the molecular profile of microglia, inducing both maladaptive and adaptive alterations in their activities. Increased expression of TREM2 seems to support microglial activities that counteract this type of network dysfunction.

Activation of the complement system propagates neuroinflammation and brain damage early and chronically after traumatic brain injury (TBI). The complement system is complex and comprises more than 50 components, many of which remain to be characterized in the normal and injured brain. Moreover, complement therapeutic studies have focused on a limited number of histopathological outcomes, which while informative, do not assess the effect of complement inhibition on neuroprotection and inflammation in a comprehensive manner. Using high throughput gene expression technology (NanoString), we simultaneously analyzed complement gene expression profiles with other neuroinflammatory pathway genes at different time points after TBI. We additionally assessed the effects of complement inhibition on neuropathological processes. Analyses of neuroinflammatory genes were performed at days 3, 7, and 28 post injury in male C57BL/6 mice following a controlled cortical impact injury. We also characterized the expression of 59 complement genes at similar time points, and also at 1- and 2-years post injury. Overall, TBI upregulated the expression of markers of astrogliosis, immune cell activation, and cellular stress, and downregulated the expression of neuronal and synaptic markers from day 3 through 28 post injury. Moreover, TBI upregulated gene expression across most complement activation and effector pathways, with an early emphasis on classical pathway genes and with continued upregulation of C2, C3 and C4 expression 2 years post injury. Treatment using the targeted complement inhibitor, CR2-Crry, significantly ameliorated TBI-induced transcriptomic changes at all time points. Nevertheless, some immune and synaptic genes remained dysregulated with CR2-Crry treatment, suggesting adjuvant anti-inflammatory and neurotropic therapy may confer additional neuroprotection. In addition to characterizing complement gene expression in the normal and aging brain, our results demonstrate broad and chronic dysregulation of the complement system after TBI, and strengthen the view that the complement system is an attractive target for TBI therapy.

METHODS : Retinal, optic nerve head (ONH) and distal optic nerve (ON) tissues from 8 juvenile 10-12 week-old cats (4 males and 4 females) with feline congenital glaucoma (FCG) and 5 age-matched normal control cats (3 males and 2 females) were used. Data for weekly intraocular pressure (IOP) and optic nerve axon counts were available for all subjects. Protein and gene expression in tissue cryosections were examined by immunofluorescence labeling (IF) and RNAscope in situ hybridization (ISH), respectively. Retinal tissue was IF labeled for myeloid cell marker, IBA-1 and flat-mounted. ISH for markers of infiltrating monocytes/macrophages (_CCR2_) and proinflammatory cytokines (_IL1A_, _C1QA_, _TNF_) was performed. Microglia were identified by IF of homeostatic microglial marker, P2RY12. Microscopy images wereanalyzed using Image J, QuPath and Imaris. Two-tailed unpaired t-test or Mann-Whitney test or ANOVA were used for between-group comparisons (p