Sex-distinct microglial activation and myeloid cell infiltration in the spinal cord after painful peripheral injury.

Chronic pain is a common and often debilitating problem that affects 100 million Americans. A better understanding of pain's molecular mechanisms is necessary for developing safe and effective therapeutics. Microglial activation has been implicated as a mediator of chronic pain in numerous preclinical studies; unfortunately, translational efforts using known glial modulators have largely failed, perhaps at least in part due to poor specificity of the compounds pursued, or an incomplete understanding of microglial reactivity. In order to achieve a more granular understanding of the role of microglia in chronic pain as a means of optimizing translational efforts, we utilized a clinically-informed mouse model of complex regional pain syndrome (CRPS), and monitored microglial activation throughout pain progression. We discovered that while both males and females exhibit spinal cord microglial activation as evidenced by increases in Iba1, activation is attenuated and delayed in females. We further evaluated the expression of the newly identified microglia-specific marker, TMEM119, and identified two distinct populations in the spinal cord parenchyma after peripheral injury: TMEM119+ microglia and TMEM119- infiltrating myeloid lineage cells, which are comprised of Ly6G + neutrophils and Ly6G- macrophages/monocytes. Neurons are sensitized by inflammatory mediators released in the CNS after injury; however, the cellular source of these cytokines remains somewhat unclear. Using multiplex in situ hybridization in combination with immunohistochemistry, we demonstrate that spinal cord TMEM119+ microglia are the cellular source of cytokines IL6 and IL1ß after peripheral injury. Taken together, these data have important implications for translational studies: 1) microglia remain a viable analgesic target for males and females, so long as duration after injury is considered; 2) the analgesic properties of microglial modulators are likely at least in part related to their suppression of microglial-released cytokines, and 3) a limited number of neutrophils and macrophages/monocytes infiltrate the spinal cord after peripheral injury but have unknown impact on pain persistence or resolution. Further studies to uncover glial-targeted therapeutic interventions will need to consider sex, timing after injury, and the exact target population of interest to have the specificity necessary for translation.

NAD+ supplementation reduces neuroinflammation and cell senescence in a transgenic mouse model of Alzheimer's disease via cGAS-STING.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder. Impaired neuronal bioenergetics and neuroinflammation are thought to play key roles in the progression of AD, but their interplay is not clear. Nicotinamide adenine dinucleotide (NAD+) is an important metabolite in all human cells in which it is pivotal for multiple processes including DNA repair and mitophagy, both of which are impaired in AD neurons. Here, we report that levels of NAD+ are reduced and markers of inflammation increased in the brains of APP/PS1 mutant transgenic mice with beta-amyloid pathology. Treatment of APP/PS1 mutant mice with the NAD+ precursor nicotinamide riboside (NR) for 5 mo increased brain NAD+ levels, reduced expression of proinflammatory cytokines, and decreased activation of microglia and astrocytes. NR treatment also reduced NLRP3 inflammasome expression, DNA damage, apoptosis, and cellular senescence in the AD mouse brains. Activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are associated with DNA damage and senescence. cGAS-STING elevation was observed in the AD mice and normalized by NR treatment. Cell culture experiments using microglia suggested that the beneficial effects of NR are, in part, through a cGAS-STING-dependent pathway. Levels of ectopic (cytoplasmic) DNA were increased in APP/PS1 mutant mice and human AD fibroblasts and down-regulated by NR. NR treatment induced mitophagy and improved cognitive and synaptic functions in APP/PS1 mutant mice. Our findings suggest a role for NAD+ depletion-mediated activation of cGAS-STING in neuroinflammation and cellular senescence in AD.

Longitudinal translocator protein-18 kDa-positron emission tomography imaging of peripheral and central myeloid cells in a mouse model of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a severely disabling disease characterized by pain, temperature changes, motor dysfunction, and edema that most often occurs as an atypical response to a minor surgery or fracture. Inflammation involving activation and recruitment of innate immune cells, including both peripheral and central myeloid cells (ie, macrophages and microglia, respectively), is a key feature of CRPS. However, the exact role and time course of these cellular processes relative to the known acute and chronic phases of the disease are not fully understood. Positron emission tomography (PET) of translocator protein-18 kDa (TSPO) is a method for noninvasively tracking these activated innate immune cells. Here, we reveal the temporal dynamics of peripheral and central inflammatory responses over 20 weeks in a tibial fracture/casting mouse model of CRPS through longitudinal TSPO-PET using [F]GE-180. Positron emission tomography tracer uptake quantification in the tibia revealed increased peripheral inflammation as early as 2 days after fracture and lasting 7 weeks. Centralized inflammation was detected in the spinal cord and brain of fractured mice at 7 and 21 days after injury. Spinal cord tissue immunofluorescent staining revealed TSPO expression in microglia (CD11b+) at 7 days but was restricted mainly to endothelial cells (PECAM1+) at baseline and 7 weeks. Our data suggest early and persistent peripheral myeloid cell activation and transient central microglial activation are limited to the acute phase of CRPS. Moreover, we show that TSPO-PET can be used to noninvasively monitor the spatiotemporal dynamics of myeloid cell activation in CRPS progression with potential to inform disease phase-specific therapeutics.

Microglial Modulation as a Target for Chronic Pain: From the Bench to the Bedside and Back.

With a widespread opioid epidemic and profound biopsychosocial implications, chronic pain is a multifaceted public health issue requiring urgent attention. The treatment of chronic pain is particularly important to anesthesiologists given our unique role as perioperative physicians and pain medicine specialists. The present review details the recent shift from a neuronal theory of chronic pain to one that includes complex neuron-glia interactions. In particular, we highlight microglia, the myeloid-lineage cells of the central nervous system, as initiators of a postinjury neuroimmune response that contributes to the acute to chronic pain transition. We discuss ever-advancing preclinical studies, wherein significant success has been made through pharmacologic and genetic modulation of microglia, and we emphasize where these approaches have made the transition to the clinical realm. Furthermore, we highlight the most current, novel efforts to visualize glial activation in vivo using positron emission tomography and improve the diagnosis of chronic pain through radiotracer binding of specific targets, like the 18 kDa translocator protein in microglia and myeloid-lineage cells. Our rapidly advancing knowledge about microglia and their involvement in pain suggests that the era of glial-targeted therapeutics is just beginning so long as we refocus our attention on optimizing preclinical studies using a clinically informed approach, before translation.

NAD+ supplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency.

Emerging findings suggest that compromised cellular bioenergetics and DNA repair contribute to the pathogenesis of Alzheimer's disease (AD), but their role in disease-defining pathology is unclear. We developed a DNA repair-deficient 3xTgAD/Polß+/- mouse that exacerbates major features of human AD including phosphorylated Tau (pTau) pathologies, synaptic dysfunction, neuronal death, and cognitive impairment. Here we report that 3xTgAD/Polß+/- mice have a reduced cerebral NAD+/NADH ratio indicating impaired cerebral energy metabolism, which is normalized by nicotinamide riboside (NR) treatment. NR lessened pTau pathology in both 3xTgAD and 3xTgAD/Polß+/- mice but had no impact on amyloid ß peptide (Aß) accumulation. NR-treated 3xTgAD/Polß+/- mice exhibited reduced DNA damage, neuroinflammation, and apoptosis of hippocampal neurons and increased activity of SIRT3 in the brain. NR improved cognitive function in multiple behavioral tests and restored hippocampal synaptic plasticity in 3xTgAD mice and 3xTgAD/Polß+/- mice. In general, the deficits between genotypes and the benefits of NR were greater in 3xTgAD/Polß+/- mice than in 3xTgAD mice. Our findings suggest a pivotal role for cellular NAD+ depletion upstream of neuroinflammation, pTau, DNA damage, synaptic dysfunction, and neuronal degeneration in AD. Interventions that bolster neuronal NAD+ levels therefore have therapeutic potential for AD.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

DNA polymerase ß decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase-ß (Polß) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polß levels. Compared to wild-type control mice, Polß heterozygous (Polß+/- ), and 3xTgAD mice, 3xTgAD/Polß+/- mice exhibited impaired performance in a buried food test of olfaction. Polß deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polß+/- and 3xTgAD mice, and by over 60% in the 3xTgAD/Polß+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polß+/- mice compared to 3xTgAD mice. Levels of amyloid ß-peptide (Aß) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polß+/- mice, and cultured Polß-deficient neurons exhibited increased vulnerability to Aß-induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.