Native-state proteomics of Parvalbumin interneurons identifies unique molecular signatures and vulnerabilities to early Alzheimer's pathology.

Dysfunction in fast-spiking parvalbumin interneurons (PV-INs) may represent an early pathophysiological perturbation in Alzheimer's Disease (AD). Defining early proteomic alterations in PV-INs can provide key biological and translationally-relevant insights. We used cell-type-specific in-vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state PV-IN proteomes. PV-IN proteomic signatures include high metabolic and translational activity, with over-representation of AD-risk and cognitive resilience-related proteins. In bulk proteomes, PV-IN proteins were associated with cognitive decline in humans, and with progressive neuropathology in humans and the 5xFAD mouse model of Aß pathology. PV-IN CIBOP in early stages of Aß pathology revealed signatures of increased mitochondria and metabolism, synaptic and cytoskeletal disruption and decreased mTOR signaling, not apparent in whole-brain proteomes. Furthermore, we demonstrated pre-synaptic defects in PV-to-excitatory neurotransmission, validating our proteomic findings. Overall, in this study we present native-state proteomes of PV-INs, revealing molecular insights into their unique roles in cognitive resiliency and AD pathogenesis.

Profound seizure suppression and disease modification by targeting JAK1, a key driver of a pro-epileptogenic gene network.

Epilepsy is the 4th most prevalent neurological disorder with over 50 million cases worldwide. While a number of drugs exist to suppress seizures, approximately 1/3 of patients remain drug resistant, and no current treatments are disease modifying. Using network and systems-based approaches, we find that the histone methylase EZH2 suppresses epileptogenesis and slows disease progression, via repression of JAK1 and STAT3 signaling in hippocampal neurons. Pharmacological inhibition of JAK1 with the orally available, FDA-approved drug CP690550 (Tofacitinib) profoundly suppresses behavioral and electrographic seizures after the onset of epilepsy across preclinical rodent models of acquired epilepsy. This seizure suppression persists for weeks after drug withdrawal. Identification of an endogenous protective response to status epilepticus in the form of EZH2 induction has highlighted a critical role for the JAK1 kinase and STAT3 in both the initiation and propagation of epilepsy across preclinical rodent models and human disease. Overall, we find that STAT3 is transiently activated after insult, reactivates with spontaneous seizures, and remains targetable for disease modification in chronic epilepsy.

Native-state proteomics of Parvalbumin interneurons identifies novel molecular signatures and metabolic vulnerabilities to early Alzheimer's disease pathology.

One of the earliest pathophysiological perturbations in Alzheimer's Disease (AD) may arise from dysfunction of fast-spiking parvalbumin (PV) interneurons (PV-INs). Defining early protein-level (proteomic) alterations in PV-INs can provide key biological and translationally relevant insights. Here, we use cell-type-specific in vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state proteomes of PV interneurons. PV-INs exhibited proteomic signatures of high metabolic, mitochondrial, and translational activity, with over-representation of causally linked AD genetic risk factors. Analyses of bulk brain proteomes indicated strong correlations between PV-IN proteins with cognitive decline in humans, and with progressive neuropathology in humans and mouse models of Aß pathology. Furthermore, PV-IN-specific proteomes revealed unique signatures of increased mitochondrial and metabolic proteins, but decreased synaptic and mTOR signaling proteins in response to early Aß pathology. PV-specific changes were not apparent in whole-brain proteomes. These findings showcase the first native state PV-IN proteomes in mammalian brain, revealing a molecular basis for their unique vulnerabilities in AD.

Preclinical development of an EP2 antagonist for post-seizure cognitive deficits.

Cognitive comorbidities can substantially reduce quality of life in people with epilepsy. Inflammation is a component of all chronic diseases including epilepsy, as well as acute events like status epilepticus (SE). Neuroinflammation is the consequence of several broad signaling cascades including cyclooxygenase-2 (COX-2)-associated pathways. Activation of the EP2 receptor for prostaglandin E2 appears responsible for blood-brain barrier leakage and much of the inflammatory reaction, neuronal injury and cognitive deficit that follows seizure-provoked COX-2 induction in brain. Here we show that brief exposure of mice to TG11-77, a potent, selective, orally available and brain permeant EP2 antagonist, eliminates the profound cognitive deficit in Y-maze performance after SE and reduces delayed mortality and microgliosis, with a minimum effective i.p. dose (as free base) of 8.8 mg/kg. All in vitro studies required to submit an investigational new drug (IND) application for TG11-77 have been completed, and non-GLP dose range-finding toxicology in the rat identified no overt, organ or histopathology signs of toxicity after 7 days of oral administration at 1000 mg/kg/day. Plasma exposure in the rat was dose-linear between 15 and 1000 mg/kg dosing. TG11-77 thus appears poised to continue development towards the initial clinical test of the hypothesis that EP2 receptor modulation after SE can provide the first preventive treatment for one of the chief comorbidities of epilepsy.

Sleep Disruption Worsens Seizures: Neuroinflammation as a Potential Mechanistic Link.

Sleep disturbances, such as insomnia, obstructive sleep apnea, and daytime sleepiness, are common in people diagnosed with epilepsy. These disturbances can be attributed to nocturnal seizures, psychosocial factors, and/or the use of anti-epileptic drugs with sleep-modifying side effects. Epilepsy patients with poor sleep quality have intensified seizure frequency and disease progression compared to their well-rested counterparts. A better understanding of the complex relationship between sleep and epilepsy is needed, since approximately 20% of seizures and more than 90% of sudden unexpected deaths in epilepsy occur during sleep. Emerging studies suggest that neuroinflammation, (e.g., the CNS immune response characterized by the change in expression of inflammatory mediators and glial activation) may be a potential link between sleep deprivation and seizures. Here, we review the mechanisms by which sleep deprivation induces neuroinflammation and propose that neuroinflammation synergizes with seizure activity to worsen neurodegeneration in the epileptic brain. Additionally, we highlight the relevance of sleep interventions, often overlooked by physicians, to manage seizures, prevent epilepsy-related mortality, and improve quality of life.

Impact of Stress on Epilepsy: Focus on Neuroinflammation-A Mini Review.

Epilepsy, one of the most common neurological disorders worldwide, is characterized by recurrent seizures and subsequent brain damage. Despite strong evidence supporting a deleterious impact on seizure occurrence and outcome severity, stress is an overlooked component in people with epilepsy. With regard to stressor duration and timing, acute stress can be protective in epileptogenesis, while chronic stress often promotes seizure occurrence in epilepsy patients. Preclinical research suggests that chronic stress promotes neuroinflammation and leads to a depressive state. Depression is the most common psychiatric comorbidity in people with epilepsy, resulting in a poor quality of life. Here, we summarize studies investigating acute and chronic stress as a seizure trigger and an important factor that worsens epilepsy outcomes and psychiatric comorbidities. Mechanistic insight into the impact of stress on epilepsy may create a window of opportunity for future interventions targeting neuroinflammation-related disorders.

Progesterone Modulates Mitochondrial Functions in Human Glioblastoma Cells.

A substantial literature supports the notion that cancer is a metabolic disease. Mitochondria are sexually dimorphic, and progesterone (P4) plays a key regulatory role in mitochondrial functions. We investigated the effect of P4 on mitochondrial functions in three human glioblastoma multiforme (GBM) cell lines. In dose-response and time-response studies, GBM cells were exposed to different concentrations of P4 followed by mitochondrial stress-testing with a Seahorse analyzer. Data were analyzed for oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and spare respiratory capacity (SRC) to determine the effects of P4 exposure on mitochondrial respiration and rate of glycolysis. We also examined the effect of P4 on mitochondrial superoxide radical generation by confocal microscopy. As early as 1h post-P4 exposure, we found a substantial dose-dependent inhibitory effect of P4 on OCR, ECAR, and SRC in all GBM cell lines. P4 treatment altered the levels of basal respiration, maximum respiration, nonmitochondrial oxygen consumption, ATP production, and proton leak. P4 given at 80-µM concentration showed the maximum inhibitory effect compared to controls. Live imaging data showed an 11-22% increase in superoxide radical generation in all three GBM cell lines following 6h exposure to a high concentration of P4. Our data show that high-dose P4 exerts an inhibitory effect on both mitochondrial respiration and glycolysis in GBM cells. These effects would lead to decreased tumor size and rate of growth, representing a potential treatment to control the spread of GBM.

Peripheral Myeloid Cell EP2 Activation Contributes to the Deleterious Consequences of Status Epilepticus.

A multidimensional inflammatory response ensues after status epilepticus (SE), driven partly by cyclooxygenase-2-mediated activation of prostaglandin EP2 receptors. The inflammatory response is typified by astrocytosis, microgliosis, erosion of the blood-brain barrier (BBB), formation of inflammatory cytokines, and brain infiltration of blood-borne monocytes. Our previous studies have shown that inhibition of monocyte brain invasion or systemic administration of an EP2 receptor antagonist relieves multiple deleterious consequences of SE. Here we identify those effects of EP2 antagonism that are reproduced by conditional ablation of EP2 receptors in immune myeloid cells and show that systemic EP2 antagonism blocks monocyte brain entry in male mice. The induction of hippocampal IL-6 after pilocarpine SE was nearly abolished in EP2 conditional KO mice. Serum albumin levels in the cortex, a measure of BBB breakdown, were significantly higher after SE in EP2-sufficient mice but not in EP2 conditional KOs. EP2 deficiency in innate immune cells accelerated the recovery from sickness behaviors following SE. Surprisingly, neurodegeneration was not alleviated in myeloid conditional KOs. Systemic EP2 antagonism prevented monocyte brain infiltration and provided broader rescue of SE-induced effects than myeloid EP2 ablation, including neuroprotection and broader suppression of inflammatory mediators. Reporter expression indicated that the cellular target of CD11b-driven Cre was circulating myeloid cells but, unexpectedly, not microglia. These findings indicate that activation of EP2 receptors on immune myeloid cells drives substantial deficits in behavior and disrupts the BBB after SE. The benefits of systemic EP2 antagonism can be attributed, in part, to blocking brain recruitment of blood-borne monocytes.SIGNIFICANCE STATEMENT Unabated seizures reduce quality of life, promote the development of epilepsy, and can be fatal. We previously identified activation of prostaglandin EP2 receptors as a driver of undesirable consequences of seizures. However, the relevant EP2-expressing cell types remain unclear. Here we identify peripheral innate immune cells as a driver of the EP2-related negative consequences of seizures. Removal of EP2 from peripheral immune cells was beneficial, abolishing production of a key inflammatory cytokine, accelerating weight regain, and limiting behavioral deficits. These findings provide evidence that EP2 engagement on peripheral immune and brain endothelia contributes to the deleterious effects of SE, and will assist in the development of beneficial therapies to enhance quality of life in individuals who suffer prolonged seizures.

Stroke-Induced Peripheral Immune Dysfunction in Vitamin D-Deficient Conditions: Modulation by Progesterone and Vitamin D.

Vitamin D deficiency (Ddef) alters morphology and outcomes after a stroke. We investigated the interaction of Ddef following post-stroke systemic inflammation and evaluated whether administration of progesterone (P) or vitamin D (D) will improve outcomes. Ddef rats underwent stroke with lipopolysaccharide (LPS)-induced systemic inflammation. Rats were randomly divided into 9 groups and treated with P, D, or vehicle for 4 days. At day 4, rats were tested on different behavioral parameters. Markers of neuronal inflammation, endoplasmic reticulum stress, oxidative stress, white matter integrity, and apoptosis were measured along with immune cell populations from the spleen, thymus, and blood. Severely altered outcomes were observed in the Ddef group compared to the D-sufficient (Dsuf) group. Stroke caused peripheral immune dysfunction in the Dsuf group which was worse in the Ddef group. Systemic inflammation exacerbated injury outcomes in the Dsuf group and these were worse in the Ddef group. Monotherapy with P/D showed beneficial functional effects but the combined treatment showed better outcomes than either alone. Ddef as a comorbid condition with stroke worsens stroke outcomes and can delay functional recovery. Combination treatment with P and D might be promising for future stroke therapeutics in Ddef.

Post-ischemic stroke systemic inflammation: Immunomodulation by progesterone and vitamin D hormone.

Post-stroke systemic inflammation, due to the injury itself and exacerbated by in-hospital infections, can increase morbidity and mortality in stroke patients. In this study, we examined the immunomodulatory effects of progesterone (P4) alone and in combination with vitamin D hormone (VDH) on acute phase post-stroke peripheral immune dysfunction and functional/behavioral deficits. Adult rats underwent transient middle cerebral artery occlusion/reperfusion (tMCAO) and delayed systemic inflammation was induced by injections of lipopolysaccharide (LPS) beginning 24 h post-stroke. Animals were tested for behavioral outcomes and immune function at day 4 post-stroke. We also measured infarction volume and markers of neuronal inflammation (GFAP, IL-6) and apoptosis (cleaved caspase-3) in brain post-stroke. We observed the worst stroke outcomes in the stroke + systemic inflammation group compared to the stroke-alone group. Flow cytometric analysis of different subsets of immune cells in blood, spleen and thymus revealed peripheral immune dysfunction which was restored by both P4 and VDH monotherapy. P4 monotherapy reduced infarction volume, behavioral/functional deficits, peripheral immune dysfunction, neuronal inflammation, and apoptosis induced by post-stroke systemic inflammation. Combination treatment with P4+VDH improved outcomes better than monotherapy. Our findings can be taken to suggest that the current standard of care for stroke and post-stroke infection can be substantially improved by P4 and VDH combination therapy.

Progesterone Attenuates Stress-Induced NLRP3 Inflammasome Activation and Enhances Autophagy following Ischemic Brain Injury.

NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome inhibition and autophagy induction attenuate inflammation and improve outcome in rodent models of cerebral ischemia. However, the impact of chronic stress on NLRP3 inflammasome and autophagic response to ischemia remains unknown. Progesterone (PROG), a neuroprotective steroid, shows promise in reducing excessive inflammation associated with poor outcome in ischemic brain injury patients with comorbid conditions, including elevated stress. Stress primes microglia, mainly by the release of alarmins such as high-mobility group box-1 (HMGB1). HMGB1 activates the NLRP3 inflammasome, resulting in pro-inflammatory interleukin (IL)-1ß production. In experiment 1, adult male Sprague-Dawley rats were exposed to social defeat stress for 8 days and then subjected to global ischemia by the 4-vessel occlusion model, a clinically relevant brain injury associated with cardiac arrest. PROG was administered 2 and 6 h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia. Here, we show that stress and global ischemia exert a synergistic effect in HMGB1 release, resulting in exacerbation of NLRP3 inflammasome activation and autophagy impairment in the hippocampus of ischemic animals. In experiment 2, an in vitro inflammasome assay, primary microglia isolated from neonatal brain tissue, were primed with lipopolysaccharide (LPS) and stimulated with adenosine triphosphate (ATP), displaying impaired autophagy and increased IL-1ß production. In experiment 3, hippocampal microglia isolated from stressed and unstressed animals, were stimulated ex vivo with LPS, exhibiting similar changes than primary microglia. Treatment with PROG reduced HMGB1 release and NLRP3 inflammasome activation, and enhanced autophagy in stressed and unstressed ischemic animals. Pre-treatment with an autophagy inhibitor blocked Progesterone's (PROG's) beneficial effects in microglia. Our data suggest that modulation of microglial priming is one of the molecular mechanisms by which PROG ameliorates ischemic brain injury under stressful conditions.

Sex differences in the performance of cognitive tasks in a murine model of metabolic syndrome.

The metabolic syndrome includes changes in blood glucose levels, arterial hypertension, triglycerides, dyslipidemia and central obesity. Countless reports have described the correlation between the metabolic syndrome and cognitive impairment. However, only a few reports have assessed cognitive impairment associated with the metabolic syndrome in animals of both sexes. For this purpose, Sprague-Dawley male and female rats were fed either with a hypercaloric diet as model of the metabolic syndrome or with a standard chow diet as controls. Subsequently, spatial learning and memory (Morris water maze) as well as short- and long-term memory (passive avoidance task) were evaluated. Body weight, blood pressure, triglycerides, and total cholesterol significantly increased (F(1, 36) = 94.89, p < .001) in rats fed with hypercaloric diet compared to control rats. Furthermore, cognitive impairment was observed in spatial learning and spatial memory on male rats but not on female rats fed with hypercaloric diet. In addition, a long-term memory impairment was observed in both groups fed with hypercaloric diet in comparison to their respective control group (F(1, 32) = 10.61, p = .0027). Immunohistochemistry results showed no changes in the number of positive cells for NeuN, GFAP and Ox-42. In males fed with a hypercaloric diet, a decrease in testosterone levels was observed, whereas estradiol levels decreased in females when compared with their respective control group (p < .0001). In this MetS animal model, metabolic and cognitive differences were observed in males and females, which demonstrates that sex hormones play a significant role in metabolic regulation and neuroprotection related to the CA1 region of the hippocampus.

Progesterone Treatment Attenuates Glycolytic Metabolism and Induces Senescence in Glioblastoma.

We examined the effect of progesterone treatments on glycolytic metabolism and senescence as possible mechanisms in controlling the growth of glioblastoma multiforme (GBM). In an orthotopic mouse model, after tumor establishment, athymic nude mice received treatment with progesterone or vehicle for 40 days. Compared to controls, high-dose progesterone administration produced a significant reduction in tumor size (~47%) and an increased survival rate (~43%) without any demonstrable toxicity to peripheral organs (liver, kidney). This was accompanied by a significant improvement in spontaneous locomotor activity and reduced anxiety-like behavior. In a follow-up in vitro study of U87MG-luc, U87dEGFR and U118MG tumor cells, we observed that high-dose progesterone inhibited expression of Glut1, which facilitated glucose transport into the cytoplasm; glyceraldehyde 3-phosphate dehydrogenase (GAPDH; a glycolysis enzyme); ATP levels; and cytoplasmic FoxO1 and Phospho-FoxO1, both of which control glycolytic metabolism through upstream PI3K/Akt/mTOR signaling in GBM. In addition, progesterone administration attenuated EGFR/PI3K/Akt/mTOR signaling, which is highly activated in grade IV GBM. High-dose progesterone also induced senescence in GBM as evidenced by changes in cell morphology and ß-galactocidase accumulation. In conclusion, progesterone inhibits the modulators of glycolytic metabolism and induces premature senescence in GBM cells and this can help to reduce/slow tumor progression.

Neurosteroid allopregnanolone reduces ipsilateral visual cortex potentiation following unilateral optic nerve injury.

In adult mice with unilateral optic nerve crush injury (ONC), we studied visual response plasticity in the visual cortex following stimulation with sinusoidal grating. We examined visually evoked potentials (VEP) in the primary visual cortex ipsilateral and contralateral to the crushed nerve. We found that unilateral ONC induces enhancement of visual response on the side ipsilateral to the injury that is evoked by visual stimulation to the intact eye. This enhancement was associated with supranormal spatial frequency thresholds in the intact eye when tested using optomotor response. To probe whether injury-induced disinhibition caused the potentiation, we treated animals with the neurosteroid allopregnanolone, a potent agonist of the GABAA receptor, one hour after crush and on post-injury days 3, 8, 13, and 18. Allopregnanolone diminished enhancement of the VEP and this effect was associated with the upregulated synthesis of the δ-subunit of the GABAA receptor. Our study shows a new aspect of experience-dependent plasticity following unilateral ONC. This hyper-activity in the ipsilateral visual cortex is prevented by upregulation of GABA inhibition with allopregnanolone. Our findings suggest the therapeutic potential of allopregnanolone for modulation of plasticity in certain eye and brain disorders and a possible role for disinhibition in ipsilateral hyper-activity following unilateral ONC.

Stress primes microglial polarization after global ischemia: Therapeutic potential of progesterone.

Despite the fact that stress is associated with increased risk of stroke and worsened outcome, most preclinical studies have ignored this comorbid factor, especially in the context of testing neuroprotective treatments. Preclinical research suggests that stress primes microglia, resulting in an enhanced reactivity to a subsequent insult and potentially increasing vulnerability to stroke. Ischemia-induced activated microglia can be polarized into a harmful phenotype, M1, which produces pro-inflammatory cytokines, or a protective phenotype, M2, which releases anti-inflammatory cytokines and neurotrophic factors. Selective modulation of microglial polarization by inhibiting M1 or stimulating M2 may be a potential therapeutic strategy for treating cerebral ischemia. Our laboratory and others have shown progesterone to be neuroprotective against ischemic stroke in rodents, but it is not known whether it will be as effective under a comorbid condition of chronic stress. Here we evaluated the neuroprotective effect of progesterone on the inflammatory response in the hippocampus after exposure to stress followed by global ischemia. We focused on the effects of microglial M1/M2 polarization and pro- and anti-inflammatory mediators in stressed ischemic animals. Male Sprague-Dawley rats were exposed to 8 consecutive days of social defeat stress and then subjected to global ischemia or sham surgery. The rats received intraperitoneal injections of progesterone (8mg/kg) or vehicle at 2h post-ischemia followed by subcutaneous injections at 6h and once every 24h post-injury for 7days. The animals were killed at 7 and 14days post-ischemia, and brains were removed and processed to assess outcome measures using histological, immunohistochemical and molecular biology techniques. Pre-ischemic stress (1) exacerbated neuronal loss and neurodegeneration as well as microglial activation in the selectively vulnerable CA1 hippocampal region, (2) dysregulated microglial polarization, leading to upregulation of both M1 and M2 phenotype markers, (3) increased pro-inflammatory cytokine expression, and (4) reduced anti-inflammatory cytokine and neurotrophic factor expression in the ischemic hippocampus. Treatment with progesterone significantly attenuated stress-induced microglia priming by modulating polarized microglia and the inflammatory environment in the hippocampus, the area most vulnerable to ischemic injury. Our findings can be taken to suggest that progesterone holds potential as a candidate for clinical testing in ischemic stroke where high stress may be a contributing factor.

Effects of progesterone on neurite growth inhibitors in the hippocampus following global cerebral ischemia.

In this study, the effects of progesterone (P4) on the immunoreactivity to the neurite growth inhibitor Nogo-A, its receptor (Ng-R), and its effector Rho-A in the rat hippocampus, in association with parameters of spatial learning and memory following global cerebral ischemia, were assessed. Adult male rats were subjected to global cerebral ischemia (15 min), and treated with P4 or its vehicle at 15 min, 2, 6, 24, 48 and 72 h of reperfusion. Immunoreactivity to Nogo-A, Ng-R, and Rho-A was evaluated at 24 h, 72 h or 7 d, or at 14 d of reperfusion after rats were tested in the Morris Water Maze (MWM). Global cerebral ischemia induced an increase in Nogo-A, Ng-R, and Rho-A immunoreactivities in the cell bodies of CA1 pyramidal neurons at 24h after global cerebral ischemia, peaking at 72 h, and persisting 14 d later. In addition, at 72 h, a strong immunoreactivity was observed in the hippocampal layers where dendritic arborizations of CA1 pyramidal neurons are located. Treatment with P4 reduced Nogo-A, Ng-R, and Rho-A immunoreactivities in CA1, particularly at 72 h of reperfusion. These effects of P4 were consistent with the parameters of a more efficient spatial learning and memory in the MWM, as compared to vehicle-treated rats. Overall results suggest the reduction of neurite growth inhibitory molecules Nogo-A, Ng-R, and Rho-A, as a part of the restorative effects of progesterone possibly allowing the plastic phenomena to occur, able to support the functional preservation of the hippocampus following global cerebral ischemia.