A Novel Flp Reporter Mouse Shows That TRPA1 Expression Is Largely Limited to Sensory Neuron Subsets.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase FlpO in TRPA1-expressing cells. We crossed the TRPA1Flp mouse with the R26ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.

Levels of Arachidonic Acid-Derived Oxylipins and Anandamide Are Elevated Among Military APOE ɛ4 Carriers With a History of Mild Traumatic Brain Injury and Post-Traumatic Stress Disorder Symptoms.

Currently approved blood biomarkers detect intracranial lesions in adult patients with mild to moderate traumatic brain injury (TBI) acutely post-injury. However, blood biomarkers are still needed to help with a differential diagnosis of mild TBI (mTBI) and post-traumatic stress disorder (PTSD) at chronic post-injury time points. Owing to the association between phospholipid (PL) dysfunction and chronic consequences of TBI, we hypothesized that examining bioactive PL metabolites (oxylipins and ethanolamides) would help identify long-term lipid changes associated with mTBI and PTSD. Lipid extracts of plasma from active-duty soldiers deployed to the Iraq/Afghanistan wars (control = 52, mTBI = 21, PTSD = 34, and TBI + PTSD = 13) were subjected to liquid chromatography/mass spectrometry analysis to examine oxylipins and ethanolamides. Linear regression analyses followed by post hoc comparisons were performed to assess the association of these lipids with diagnostic classifications. Significant differences were found in oxylipins derived from arachidonic acid (AA) between controls and mTBI, PTSD, and mTBI + PTSD groups. Levels of AA-derived oxylipins through the cytochrome P450 pathways and anandamide were significantly elevated among mTBI + PTSD patients who were carriers of the apolipoprotein E E4 allele. These studies demonstrate that AA-derived oxylipins and anandamide may be unique blood biomarkers of PTSD and mTBI + PTSD. Further, these AA metabolites may be indicative of an underlying inflammatory process that warrants further investigation. Future validation studies in larger cohorts are required to determine a potential application of this approach in providing a differential diagnosis of mTBI and PTSD in a clinical setting.

APOE ε4 and Alzheimer's disease diagnosis associated differences in L-carnitine, GBB, TMAO, and acylcarnitines in blood and brain.

BACKGROUND: The apolipoprotein E (APOE) ε4 allele, involved in fatty acid (FA) metabolism, is a major genetic risk factor for Alzheimer's disease (AD). This study examined the influence of APOE genotypes on blood and brain markers of the L-carnitine system, necessary for fatty acid oxidation (FAO), and their collective influence on the clinical and pathological outcomes of AD. METHODS: L-carnitine, its metabolites γ-butyrobetaine (GBB) and trimethylamine-n-oxide (TMAO), and its esters (acylcarnitines) were analyzed in blood from predominantly White community/clinic-based individuals (n = 372) and in plasma and brain from the Religious Order Study (ROS) (n = 79) using liquid chromatography tandem mass spectrometry (LC-MS/MS). FINDINGS: Relative to total blood acylcarnitines, levels of short chain acylcarnitines (SCAs) were higher whereas long chain acylcarnitines (LCAs) were lower in AD, which was observed pre-clinically in APOE ε4s. Plasma medium chain acylcarnitines (MCAs) were higher amongst cognitively healthy APOE ε2 carriers relative to other genotypes. Compared to their respective controls, elevated TMAO and lower L-carnitine and GBB were associated with AD clinical diagnosis and these differences were detected preclinically among APOE ε4 carriers. Plasma and brain GBB, TMAO, and acylcarnitines were also associated with post-mortem brain amyloid, tau, and cerebrovascular pathologies. INTERPRETATION: Alterations in blood L-carnitine, GBB, TMAO, and acylcarnitines occur early in clinical AD progression and are influenced by APOE genotype. These changes correlate with post-mortem brain AD and cerebrovascular pathologies. Additional studies are required to better understand the role of the FAO disturbances in AD.

Subacute and chronic proteomic and phosphoproteomic analyses of a mouse model of traumatic brain injury at two timepoints and comparison with chronic traumatic encephalopathy in human samples.

Repetitive mild traumatic brain injury (r-mTBI) is the most widespread type of brain trauma worldwide. The cumulative injury effect triggers long-lasting pathological and molecular changes that may increase risk of chronic neurodegenerative diseases. R-mTBI is also characterized by changes in the brain proteome, where the majority of molecules altered early post-TBI are different from those altered at more chronic phases. This differentiation may contribute to the heterogeneity of available data on potential therapeutic targets and may present an obstacle in developing effective treatments. Here, we aimed to characterize a proteome profile of r-mTBI in a mouse model at two time points - 3 and 24 weeks post last TBI, as this may be a more relevant therapeutic window for individuals suffering negative consequences of r-mTBI. We identified a great number of proteins and phosphoproteins that remain continuously dysregulated from 3 to 24 weeks. These proteins may serve as effective therapeutic targets for sub-acute and chronic stages of post r-mTBI. We also compared canonical pathway activation associated with either total proteins or phosphoproteins and revealed that they both are upregulated at 24 weeks. However, at 3 weeks post-TBI, only pathways associated with total proteins are upregulated, while pathways driven by phosphoproteins are downregulated. Finally, to assess the translatability of our data, we compared proteomic changes in our mouse model with those reported in autopsied human samples of Chronic Traumatic Encephalopathy (CTE) patients compared to controls. We observed 39 common proteins that were upregulated in both species and 24 common pathways associated with these proteins. These findings support the translational relevance of our mouse model of r-mTBI for successful identification and translation of therapeutic targets.

Characterization of immune profile in an aging multiple sclerosis clinic population.

BACKGROUND: There is limited data regarding adaptive immunity in older persons with Multiple Sclerosis (MS). OBJECTIVE: The aim of the present study was to quantify adaptive immune cells in younger (age less than 50) and older (age greater than 50) with MS in the context of clinical parameters (EDSS, 25-foot walk, SDMT). Subjects were either Untreated (no MS medications in 6 months), taking Injectables (interferons or glatiramer acetate), or Other approved MS treatments. RESULTS: A total of 72 subjects were enrolled (30 younger and 42 older). Older MS patients that were Untreated or taking Injectables had lower CD8 cell counts. Older MS patients demonstrated increased levels of CD4+CD25hi cells and inflammatory serum cytokines (TNF-α, IL-8). There was suggestion that MS treatments modulated IL-10. Cognition as assessed by SDMT was associated with disease duration and IL-10. CONCLUSION: Components of adaptive immunity are influenced by aging in MS which may also impact aspects of cognition as measured by SDMT.

Age and APOE affect L-carnitine system metabolites in the brain in the APOE-TR model.

With age the apolipoprotein E (APOE) E4 allele (involved in lipid homeostasis) is associated with perturbation of bioenergetics pathways in Alzheimer's disease (AD). We therefore hypothesized that in aging mice APOE genotype would affect the L-carnitine system (central to lipid bioenergetics), in the brain and in the periphery. Using liquid chromatography-mass spectrometry, levels of L-carnitine and associated metabolites: γ-butyrobetaine (GBB), crotonobetaine, as well as acylcarnitines, were evaluated at 10-, 25-, and 50-weeks, in the brain and the periphery, in a targeted replacement mouse model of human APOE (APOE-TR). Aged APOE-TR mice were also orally administered 125 mg/kg of L-carnitine daily for 7 days followed by evaluation of brain, liver, and plasma L-carnitine system metabolites. Compared to E4-TR, an age-dependent increase among E2- and E3-TR mice was detected for medium- and long-chain acylcarnitines (MCA and LCA, respectively) within the cerebrovasculature and brain parenchyma. While following L-carnitine oral challenge, E4-TR mice had higher increases in the L-carnitine metabolites, GBB and crotonobetaine in the brain and a reduction of plasma to brain total acylcarnitine ratios compared to other genotypes. These studies suggest that with aging, the presence of the E4 allele may contribute to alterations in the L-carnitine bioenergetic system and to the generation of L-carnitine metabolites that could have detrimental effects on the vascular system. Collectively the E4 allele and aging may therefore contribute to AD pathogenesis through aging-related lipid bioenergetics as well as cerebrovascular dysfunctions.

Sex-specific plasma lipid profiles of ME/CFS patients and their association with pain, fatigue, and cognitive symptoms.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex illness which disproportionally affects females. This illness is associated with immune and metabolic perturbations that may be influenced by lipid metabolism. We therefore hypothesized that plasma lipids from ME/CFS patients will provide a unique biomarker signature of disturbances in immune, inflammation and metabolic processes associated with ME/CFS. METHODS: Lipidomic analyses were performed on plasma from a cohort of 50 ME/CFS patients and 50 controls (50% males and similar age and ethnicity per group). Analyses were conducted with nano-flow liquid chromatography (nLC) and high-performance liquid chromatography (HPLC) systems coupled with a high mass accuracy ORBITRAP mass spectrometer, allowing detection of plasma lipid concentration ranges over three orders of magnitude. We examined plasma phospholipids (PL), neutral lipids (NL) and bioactive lipids in ME/CFS patients and controls and examined the influence of sex on the relationship between lipids and ME/CFS diagnosis. RESULTS: Among females, levels of total phosphatidylethanolamine (PE), omega-6 arachidonic acid-containing PE, and total hexosylceramides (HexCer) were significantly decreased in ME/CFS compared to controls. In males, levels of total HexCer, monounsaturated PE, phosphatidylinositol (PI), and saturated triglycerides (TG) were increased in ME/CFS patients compared to controls. Additionally, omega-6 linoleic acid-derived oxylipins were significantly increased in male ME/CFS patients versus male controls. Principal component analysis (PCA) identified three major components containing mostly PC and a few PE, PI and SM species-all of which were negatively associated with headache and fatigue severity, irrespective of sex. Correlations of oxylipins, ethanolamides and ME/CFS symptom severity showed that lower concentrations of these lipids corresponded with an increase in the severity of headaches, fatigue and cognitive difficulties and that this association was influenced by sex. CONCLUSION: The observed sex-specific pattern of dysregulated PL, NL, HexCer and oxylipins in ME/CFS patients suggests a possible role of these lipids in promoting immune dysfunction and inflammation which may be among the underlying factors driving the clinical presentation of fatigue, chronic pain, and cognitive difficulties in ill patients. Further evaluation of lipid metabolism pathways is warranted to better understand ME/CFS pathogenesis.

Candida rugosa lipase alters the gastrointestinal environment in wild-type mice.

Diet and commercially available supplements can significantly impact the gut microbial composition; however, the effects of supplements often lack scientific data demonstrating the effects on healthy and diseased individuals. Hence, it was investigated, whether a frequently used supplement in humans, Candida rugosa lipase (CRL), gets delivered active beyond the stomach in the intestinal tract of C57BL/6 J mice and its impact on the gut microbial community and environment. We showed for the first time the movement of CRL in an active state through the mouse digestive tract by determination of intestinal CRL activity and free fatty acids concentrations. The short- and long-term administration of CRL resulted in significant alterations of the gut microbiome, favoring the growth of, for instance, Verrucomicrobia but also other species associated with normal body mass index (BMI) or butyrate expression, both considered beneficial. In addition, we showed that these changes persisted after supplementation and that gut barrier integrity was unaffected by the treatment. In conclusion, CRL can be delivered in an active state beyond the stomach and supplementation altered the murine gut microbiome favoring beneficial bacterial species, which may be of relevance in humans in healthy but also potentially in disease states.

Targeting sirtuin activity with nicotinamide riboside reduces neuroinflammation in a GWI mouse model.

Gulf War Illness (GWI) affects 30% of veterans from the 1991 Gulf War (GW), who suffer from symptoms that reflect ongoing mitochondria dysfunction. Brain mitochondria bioenergetics dysfunction in GWI animal models corresponds with astroglia activation and neuroinflammation. In a pilot study of GW veterans (n = 43), we observed that blood nicotinamide adenine dinucleotide (NAD) and sirtuin 1 (Sirt1) protein levels were decreased in the blood of veterans with GWI compared to healthy GW veterans. Since nicotinamide riboside (NR)-mediated targeting of Sirt1 is shown to improve mitochondria function, we tested whether NR can restore brain bioenergetics and reduce neuroinflammation in a GWI mouse model. We administered a mouse diet supplemented with NR at 100µg/kg daily for 2-months to GWI and control mice (n = 27). During treatment, mice were assessed for fatigue-type behavior using the Forced Swim Test (FST), followed by euthanasia for biochemistry and immunohistochemistry analyses. Fatigue-type behavior was elevated in GWI mice compared to control mice and lower in GWI mice treated with NR compared to untreated GWI mice. Levels of plasma NAD and brain Sirt1 were low in untreated GWI mice, while GWI mice treated with NR had higher levels, similar to those of control mice. Deacetylation of the nuclear-factor κB (NFκB) p65 subunit and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) was an increase in the brains of NR-treated GWI mice. This corresponded with a decrease in pro-inflammatory cytokines and lipid peroxidation and an increase in markers of mitochondrial bioenergetics in the brains of GWI mice. These findings suggest that targeting NR mediated Sirt1 activation restores brain bioenergetics and reduces inflammation in GWI mice. Further evaluation of NR in GWI is warranted to determine its potential efficacy in treating GWI.

Plasma Lipidomic Analyses in Cohorts With mTBI and/or PTSD Reveal Lipids Differentially Associated With Diagnosis and APOE ε4 Carrier Status.

The differential diagnosis between mild Traumatic Brain Injury (mTBI) sequelae and Post-Traumatic Stress Disorder (PTSD) is challenging due to their symptomatic overlap and co-morbidity. As such, there is a need to develop biomarkers which can help with differential diagnosis of these two conditions. Studies from our group and others suggest that blood and brain lipids are chronically altered in both mTBI and PTSD. Therefore, examining blood lipids presents a minimally invasive and cost-effective approach to identify promising biomarkers of these conditions. Using liquid chromatography-mass spectrometry (LC-MS) we examined hundreds of lipid species in the blood of healthy active duty soldiers (n = 52) and soldiers with mTBI (n = 21), PTSD (n = 34) as well as co-morbid mTBI and PTSD (n = 13) to test whether lipid levels were differentially altered with each. We also examined if the apolipoprotein E (APOE) ε4 allele can affect the association between diagnosis and peripheral lipid levels in this cohort. We show that several lipid classes are altered with diagnosis and that there is an interaction between diagnosis and the ε4 carrier status on these lipids. Indeed, total lipid levels as well as both the degree of unsaturation and chain lengths are differentially altered with diagnosis and ε4 status, specifically long chain unsaturated triglycerides (TG) and both saturated and mono-unsaturated diglycerides (DG). Additionally, an examination of lipid species reveals distinct profiles in each diagnostic group stratified by ε4 status, mainly in TG, saturated DG species and polyunsaturated phosphatidylserines. In summary, we show that peripheral lipids are promising biomarker candidates to assist with the differential diagnosis of mTBI and PTSD. Further, ε4 carrier status alone and in interaction with diagnosis has a strong influence on peripheral lipid levels. Therefore, examining ε4 status along with peripheral lipid levels could help with differential diagnosis of mTBI and PTSD.

A permethrin metabolite is associated with adaptive immune responses in Gulf War Illness.

Gulf War Illness (GWI), affecting 30% of veterans from the 1991 Gulf War (GW), is a multi-symptom illness with features similar to those of patients with autoimmune diseases. The objective of the current work is to determine if exposure to GW-related pesticides, such as permethrin (PER), activates peripheral and central nervous system (CNS) adaptive immune responses. In the current study, we focused on a PER metabolite, 3-phenoxybenzoic acid (3-PBA), as this is a common metabolite previously shown to form adducts with endogenous proteins. We observed the presence of 3-PBA and 3-PBA modified lysine of protein peptides in the brain, blood and liver of pyridostigmine bromide (PB) and  PER (PB+PER) exposed mice at acute and chronic post-exposure timepoints. We tested whether 3-PBA-haptenated albumin (3-PBA-albumin) can activate immune cells since it is known that chemically haptenated proteins can stimulate immune responses. We detected autoantibodies against 3-PBA-albumin in plasma from PB + PER exposed mice and veterans with GWI at chronic post-exposure timepoints. We also observed that in vitro treatment of blood with 3-PBA-albumin resulted in the activation of B- and T-helper lymphocytes and that these immune cells were also increased in blood of PB + PER exposed mice and veterans with GWI. These immune changes corresponded with elevated levels of infiltrating monocytes in the brain and blood of PB + PER exposed mice which coincided with alterations in the markers of blood-brain barrier disruption, brain macrophages and neuroinflammation. These studies suggest that pesticide exposure associated with GWI may have resulted in the activation of the peripheral and CNS adaptive immune responses, possibly contributing to an autoimmune-type phenotype in veterans with GWI.