Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

5-HT1F receptor agonism induces mitochondrial biogenesis and increases cellular function in brain microvascular endothelial cells.

Introduction: Vascular and mitochondrial dysfunction are well-established consequences of multiple central nervous system (CNS) disorders, including neurodegenerative diseases and traumatic injuries. We previously reported that 5-hydroxytryptamine 1F receptor (5-HT1FR) agonism induces mitochondrial biogenesis (MB) in multiple organ systems, including the CNS. Methods: Lasmiditan is a selective 5-HT1FR agonist that is FDA-approved for the treatment of migraines. We have recently shown that lasmiditan treatment induces MB, promotes vascular recovery and improves locomotor function in a mouse model of spinal cord injury (SCI). To investigate the mechanism of this effect, primary cerebral microvascular endothelial cells from C57bl/6 mice (mBMEC) were used. Results: Lasmiditan treatment increased the maximal oxygen consumption rate, mitochondrial proteins and mitochondrial density in mBMEC, indicative of MB induction. Lasmiditan also enhanced endothelial cell migration and tube formation, key components of angiogenesis. Trans-endothelial electrical resistance (TEER) and tight junction protein expression, including claudin-5, were also increased with lasmiditan, suggesting improved barrier function. Finally, lasmiditan treatment decreased phosphorylated VE-Cadherin and induced activation of the Akt-FoxO1 pathway, which decreases FoxO1-mediated inhibition of claudin-5 transcription. Discussion: These data demonstrate that lasmiditan induces MB and enhances endothelial cell function, likely via the VE-Cadherin-Akt-FoxO1-claudin-5 signaling axis. Given the importance of mitochondrial and vascular dysfunction in neuropathologies, 5-HT1FR agonism may have broad therapeutic potential to address multiple facets of disease progression by promoting MB and vascular recovery.

Serotonin regulation of mitochondria in kidney diseases.

Serotonin, while conventionally recognized as a neurotransmitter in the CNS, has recently gained attention for its role in the kidney. Specifically, serotonin is not only synthesized in the kidney, but it also regulates glomerular function, vascular resistance, and mitochondrial homeostasis. Because of serotonin's importance to mitochondrial health, this review is focused on the role of serotonin and its receptors in mitochondrial function in the context of acute kidney injury, chronic kidney disease, and diabetic kidney disease, all of which are characterized by mitochondrial dysfunction and none of which has approved pharmacological treatments. Evidence indicates that activation of certain serotonin receptors can stimulate mitochondrial biogenesis (MB) and restore mitochondrial homeostasis, resulting in improved renal function. Serotonin receptor agonists that induce MB are therefore of interest as potential therapeutic strategies for renal injury and disease. SIGNIFICANCE STATEMENT: Mitochondrial dysfunction is associated with many human renal diseases such as acute kidney injury, chronic kidney disease, and diabetic kidney disease, which are associated with increased morbidity and mortality. Unfortunately, none of these pathologies has an FDA-approved pharmacological intervention, underscoring the urgency of identifying new therapeutics for such disorders. Studies show that induction of mitochondrial biogenesis via serotonin (5-hydroxytryptamine, 5-HT) receptors reduces kidney injury markers, restores mitochondrial and renal function after kidney injury, and decreases mortality, suggesting that targeting 5-HT receptors may be a promising therapeutic avenue for mitochondrial dysfunction in kidney diseases. While numerous reviews describe the importance of mitochondria and mitochondrial quality control mechanisms in kidney disease, the relevance of 5-HT receptor-mediated mitochondrial metabolic modulation in the kidney has yet to be thoroughly explored.

Boosting Mitochondrial Biogenesis Diminishes Foam Cell Formation in the Post-Stroke Brain.

Following ischemic stroke, the degradation of myelin and other cellular membranes surpasses the lipid-processing capabilities of resident microglia and infiltrating macrophages. This imbalance leads to foam cell formation in the infarct and areas of secondary neurodegeneration, instigating sustained inflammation and furthering neurological damage. Given that mitochondria are the primary sites of fatty acid metabolism, augmenting mitochondrial biogenesis (MB) may enhance lipid processing, curtailing foam cell formation and post-stroke chronic inflammation. Previous studies have shown that the pharmacological activation of the ß2-adrenergic receptor (ß2-AR) stimulates MB. Consequently, our study sought to discern the effects of intensified ß2-AR signaling on MB, the processing of brain lipid debris, and neurological outcome using a mouse stroke model. To achieve this goal, aged mice were treated with formoterol, a long-acting ß2-AR agonist, daily for two and eight weeks following stroke. Formoterol increased MB in the infarct region, modified fatty acid metabolism, and reduced foam cell formation. However, it did not reduce markers of post-stroke neurodegeneration or improve recovery. Although our findings indicate that enhancing MB in myeloid cells can aid in the processing of brain lipid debris after stroke, it is important to note that boosting MB alone may not be sufficient to significantly impact stroke recovery.

Role of DNA methylation during recovery from spinal cord injury with and without ß2-adrenergic receptor agonism.

Daily treatment with the FDA-approved ß2-adrenergic receptor agonist formoterol beginning 8 h after severe spinal cord injury (SCI) induces mitochondrial biogenesis and improves recovery in mice. We observed decreased DNA methyltransferase (DNMT) expression, global DNA methylation and methylation of the mitochondrial genes PGC-1α and NDUFS1 in the injury site of formoterol-treated mice 1 DPI, but this effect was lost by 7 DPI. To investigate the role of DNA methylation on recovery post-SCI, injured mice were treated daily with formoterol or vehicle, plus the DNMT inhibitor decitabine (DAC) on days 7-9. While DAC had no apparent effect on formoterol-induced recovery, mice treated with vehicle plus DAC exhibited increased BMS scores compared to vehicle alone beginning 15 DPI, reaching a degree of functional recovery similar to that of formoterol-treated mice by 21 DPI. Furthermore, DAC treatment increased injury site mitochondrial protein expression in vehicle-treated mice to levels comparable to that of formoterol-treated mice. The effect of DNMT inhibition on pain response with and without formoterol was assessed following moderate SCI. While all injured mice not treated with DAC displayed thermal hyperalgesia by 21 DPI, mice treated with formoterol exhibited decreased thermal hyperalgesia compared to vehicle-treated mice by 35 DPI. Injured mice treated with DAC, regardless of formoterol treatment, did not demonstrate thermal hyperalgesia at any time point assessed. Although these data do not suggest enhanced formoterol-induced recovery with DNMT inhibition, our findings indicate the importance of DNA methylation post-SCI and support both DNMT inhibition and formoterol as potential therapeutic avenues.

FDA-approved 5-HT1F receptor agonist lasmiditan induces mitochondrial biogenesis and enhances locomotor and blood-spinal cord barrier recovery after spinal cord injury.

Vascular and mitochondrial dysfunction are well-established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. The goal of this study was to elucidate if mitochondrial biogenesis (MB) induction with a new, selective and FDA-approved 5-hydroxytryptamine receptor 1F (5-HT1F) receptor agonist, lasmiditan, can stimulate locomotor recovery and restoration of the blood-spinal cord barrier (BSCB) after SCI. Female C57BL/6 J mice were subjected to moderate SCI using a force-controlled impactor-induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1 h after injury. In the naïve spinal cord, electron microscopy revealed increased mitochondrial density and mitochondrial area, as well as enhanced mitochondrial DNA content. FCCP-uncoupled oxygen consumption rate (OCR), a functional marker of MB, was also increased in the naïve spinal cord following lasmiditan treatment. We observed disrupted mitochondrial DNA content, PGC-1α levels and FCCP-OCR in the injury site 3d after SCI. Lasmiditan treatment attenuated, and in some cases restored these deficits. Lasmiditan treatment also resulted in increased locomotor capability as early as 7d post-SCI, with treated mice reaching a Basso-Mouse Scale score of 3.3 by 21d, while vehicle-treated mice exhibited a score of 2.0. Integrity of the BSCB was assessed using Evans Blue dye extravasation. While SCI increased dye extravasation at 3d and 7d, dye accumulation in the spinal cord of lasmiditan-treated mice was attenuated 7d post-SCI, suggesting accelerated BSCB recovery. Finally, lasmiditan treatment resulted in decreased lesion volume and spared myelinated tissue 7d post-SCI. Collectively, these data reveal that 5-HT1F receptor agonist-induced MB using the FDA-approved drug lasmiditan may be an effective therapeutic strategy for the treatment of SCI.

Time-to-treatment window and cross-sex potential of ß2-adrenergic receptor-induced mitochondrial biogenesis-mediated recovery after spinal cord injury.

Mitochondrial dysfunction is a well-characterized consequence of spinal cord injury (SCI). We previously reported that treatment with the FDA-approved ß2-adrenergic receptor agonist formoterol beginning 8 h post-SCI induces mitochondrial biogenesis (MB) and improves body composition and locomotor recovery in female mice. To determine the time-to-treatment window of formoterol, female mice were subjected to 80 kdyn contusion SCI and daily administration of vehicle or formoterol (0.3 mg/kg) beginning 24 h after injury. This delayed treatment paradigm improved body composition in female mice by 21 DPI, returning body weight to pre-surgery weight and restoring gastrocnemius mass to sham levels; however, there was no effect on locomotor recovery, as measured by the Basso-Mouse Scale (BMS), or lesion volume. To assess the cross-sex potential of formoterol, injured male mice were treated with vehicle or formoterol (0.3 or 1.0 mg/kg) beginning 8 h after SCI. Formoterol also improved body composition post-SCI in male mice, restoring body weight and muscle mass regardless of dose. Interestingly, however, improved BMS scores and decreased lesion volume was observed only in male mice treated with 0.3 mg/kg. Additionally, 0.3 mg/kg formoterol induced MB in the gastrocnemius and injured spinal cord, as evidenced by increased MB protein expression and mitochondrial number. These data indicate that formoterol treatment improves recovery post-SCI in both male and female mice in a dose- and initiation time-dependent manner. Furthermore, formoterol-induced functional recovery post-SCI is not directly associated with peripheral effects, such as muscle mass and body weight.

Mitochondrial biogenesis as a therapeutic target for traumatic and neurodegenerative CNS diseases.

Central nervous system (CNS) diseases, both traumatic and neurodegenerative, are characterized by impaired mitochondrial bioenergetics and often disturbed mitochondrial dynamics. The dysregulation observed in these pathologies leads to defective respiratory chain function and reduced ATP production, thereby promoting neuronal death. As such, attenuation of mitochondrial dysfunction through induction of mitochondrial biogenesis (MB) is a promising, though still underexplored, therapeutic strategy. MB is a multifaceted process involving the integration of highly regulated transcriptional events, lipid membrane and protein synthesis/assembly and replication of mtDNA. Several nuclear transcription factors promote the expression of genes involved in oxidative phosphorylation, mitochondrial import and export systems, antioxidant defense and mitochondrial gene transcription. Of these, the nuclear-encoded peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is the most commonly studied and is widely accepted as the 'master regulator' of MB. Several recent preclinical studies document that reestablishment of mitochondrial homeostasis through increased MB results in inhibited injury progression and increased functional recovery. This perspective will briefly review the role of mitochondrial dysfunction in the propagation of CNS diseases, while also describing current research strategies that mediate mitochondrial dysfunction and compounds that induce MB for the treatment of acute and chronic neuropathologies.

Formoterol, a ß2-adrenoreceptor agonist, induces mitochondrial biogenesis and promotes cognitive recovery after traumatic brain injury.

Traumatic brain injury (TBI) leads to acute necrosis at the site of injury followed by a sequence of secondary events lasting from hours to weeks and often years. Targeting mitochondrial impairment following TBI has shown improvements in brain mitochondrial bioenergetics and neuronal function. Recently formoterol, a highly selective ß2-adrenoreceptor agonist, was found to induce mitochondrial biogenesis (MB) via Gßγ-Akt-eNOS-sGC pathway. Activation of MB is a novel approach that has been shown to restore mitochondrial function in several disease and injury models. We hypothesized that activation of MB as a target of formoterol after TBI would mitigate mitochondrial dysfunction, enhance neuronal function and improve behavioral outcomes. TBI-injured C57BL/6 male mice were injected (i.p.) with vehicle (normal saline) or formoterol (0.3 mg/kg) at 15 min, 8 h, 16 h, 24 h and then daily after controlled cortical impact (CCI) until euthanasia. After CCI, mitochondrial copy number and bioenergetic function were decreased in the ipsilateral cortex of the CCI-vehicle group. Compared to CCI-vehicle, cortical and hippocampal mitochondrial respiration rates as well as cortical mitochondrial DNA copy number were increased in the CCI-formoterol group. Mitochondrial Ca2+ buffering capacity in the hippocampus was higher in the CCI-formoterol group compared to CCI-vehicle group. Both assessments of cognitive performance, novel object recognition (NOR) and Morris water maze (MWM), decreased following CCI and were restored in the CCI-formoterol group. Although no changes were seen in the amount of cortical tissue spared between CCI-formoterol and CCI-vehicle groups, elevated levels of hippocampal neurons and improved white matter sparing in the corpus callosum were observed in CCI-formoterol group. Collectively, these results indicate that formoterol-mediated MB activation may be a potential therapeutic target to restore mitochondrial bioenergetics and promote functional recovery after TBI.

5-hydroxytryptamine 1F Receptor Agonist Induces Mitochondrial Biogenesis and Promotes Recovery from Spinal Cord Injury.

Spinal cord injury (SCI) is characterized by vascular disruption leading to ischemia, decreased oxygen delivery, and loss of mitochondrial homeostasis. This mitochondrial dysfunction results in loss of cellular functions, calcium overload, and oxidative stress. Pharmacological induction of mitochondrial biogenesis (MB) may be an effective approach to treat SCI. LY344864, a 5-hydroxytryptamine 1F (5-HT1F) receptor agonist, is a potent inducer of MB in multiple organ systems. To assess the efficacy of LY344864-induced MB on recovery post-SCI, female mice were subjected to moderate force-controlled impactor-induced contusion SCI followed by daily LY344864 administration for 21 days. Decreased mitochondrial DNA and protein content was present in the injury site 3 days post-SCI. LY344864 treatment beginning 1 h after injury attenuated these decreases, indicating MB. Additionally, injured mice treated with LY344864 displayed decreased Evan's Blue dye accumulation in the spinal cord compared with vehicle-treated mice 7 days after injury, suggesting restoration of vascular integrity. LY344864 also increased locomotor capability, with treated mice reaching a Basso-Mouse Scale score of 3.4 by 21 days, whereas vehicle-treated mice exhibited a score of 1.9. Importantly, knockout of the 5-HT1F receptor blocked LY344864-induced recovery. Remarkably, a similar degree of locomotor restoration was observed when treatment initiation was delayed until 8 h after injury. Furthermore, cross-sectional analysis of the spinal cord 21 days after injury revealed decreased lesion volume with delayed LY344864 treatment initiation, emphasizing the potential clinical applicability of this therapeutic approach. These data provide evidence that induction of MB via 5-HT1F receptor agonism may be a promising strategy for the treatment of SCI. SIGNIFICANCE STATEMENT: Treatment with LY344864 induces mitochondrial biogenesis in both the naive and injured mouse spinal cord. In addition, treatment with LY344864 beginning after impactor-induced contusion spinal cord injury improves mitochondrial homeostasis, blood-spinal cord barrier integrity, and locomotor function within 7 days. Importantly, similar locomotor results are observed whether treatment is initiated at 1 h after injury or 8 h after injury. These data indicate the potential for pharmacological induction of mitochondrial biogenesis through a 5-hydroxytryptamine 1F agonist as a novel therapeutic approach for spinal cord injury.

ß2-adrenergic receptor-mediated mitochondrial biogenesis improves skeletal muscle recovery following spinal cord injury.

In addition to local spinal cord dysfunction, spinal cord injury (SCI) can result in decreased skeletal muscle mitochondrial activity and muscle atrophy. Treatment with the FDA-approved ß2-adrenergic receptor (ADRB2) agonist formoterol has been shown to induce mitochondrial biogenesis (MB) in both the spinal cord and skeletal muscle and, therefore, has the potential to address comprehensive mitochondrial and organ dysfunction following SCI. Female C57BL/6 mice were subjected to moderate contusion SCI (80 Kdyn) followed by daily administration of vehicle or formoterol beginning 8 h after injury, a clinically relevant time-point characterized by a 50% decrease in mtDNA content in the injury site. As measured by the Basso Mouse Scale, formoterol treatment improved locomotor recovery in SCI mice compared to vehicle treatment by 7 DPI, with continued recovery observed through 21 DPI (3.5 v. 2). SCI resulted in 15% body weight loss in all mice by 3 DPI. Mice treated with formoterol returned to pre-surgery weight by 13 DPI, while no weight gain occurred in vehicle-treated SCI mice. Remarkably, formoterol-treated mice exhibited a 30% increase in skeletal muscle mass compared to those treated with vehicle 21 DPI (0.93 v. 0.72% BW), corresponding with increased MB and decreased skeletal muscle atrophy. These effects were not observed in ADRB2 knockout mice subjected to SCI, indicating that formoterol is acting via the ADRB2 receptor. Furthermore, knockout mice exhibited decreased basal spinal cord and skeletal muscle PGC-1α expression, suggesting that ADRB2 may play a role in mitochondrial homeostasis under physiological conditions. These data provide evidence for systemic ADRB2-mediated MB as a therapeutic avenue for the treatment of SCI.

Pharmacological Stimulation of Mitochondrial Biogenesis Using the Food and Drug Administration-Approved ß2-Adrenoreceptor Agonist Formoterol for the Treatment of Spinal Cord Injury.

A hallmark of the progressive cascade of damage referred to as secondary spinal cord injury (SCI) is vascular disruption resulting in decreased oxygen delivery and loss of mitochondria homeostasis. While therapeutics targeting restoration of single facets of mitochondrial function have proven largely ineffective clinically post-SCI, comprehensively addressing mitochondrial function via pharmacological stimulation of mitochondrial biogenesis (MB) is an underexplored strategy. This study examined the effects of formoterol, a mitochondrial biogenic Food and Drug Administration-approved selective and potent ß2-adrenoreceptor (ADRB2) agonist, on recovery from SCI in mice. Female C57BL/6 mice underwent moderate SCI using a force-controlled impactor-induced contusion model, followed by daily formoterol intraperitoneal administration (0.1 mg/kg) beginning 1 h post-SCI. The SCI resulted in decreased mitochondrial protein expression, including PGC-1α, in the injury and peri-injury sites as early as 3 days post-injury. Formoterol treatment attenuated this decrease in PGC-1α, indicating enhanced MB, and restored downstream mitochondrial protein expression to that of controls by 15 days. Formoterol-treated mice also exhibited less histological damage than vehicle-treated mice 3 days after injury-namely, decreased lesion volume and increased white and gray matter sparing in regions rostral and caudal to the injury epicenter. Importantly, locomotor capability of formoterol-treated mice was greater than vehicle-treated mice by 7 days, reaching a Basso Mouse Scale score two points greater than that of vehicle-treated SCI mice by 15 days. Interestingly, similar locomotor restoration was observed when initiation of treatment was delayed until 8 h post-injury. These data provide evidence of ADRB2-mediated MB as a therapeutic approach for the management of SCI.

5-HT1F receptor-mediated mitochondrial biogenesis for the treatment of Parkinson's disease.

BACKGROUND AND PURPOSE: Parkinson's disease is characterized by progressive decline in motor function due to degeneration of nigrostriatal dopaminergic neurons, as well as other deficits including cognitive impairment and behavioural abnormalities. Mitochondrial dysfunction, leading to loss of ATP-dependent cellular functions, calcium overload, excitotoxicity and oxidative stress, is implicated in the pathophysiology of Parkinson's disease. Using the 5-HT1F receptor agonist LY344864, a known inducer of mitochondrial biogenesis (MB), we investigated the therapeutic efficacy of stimulating MB on dopaminergic neuron loss in a mouse model of Parkinson's disease. EXPERIMENTAL APPROACH: Male C57BL/6 mice underwent bilateral intrastriatal 6-hydroxydopamine or saline injections and daily treatment with 2 mg·kg-1 LY344864 or vehicle for 14 days beginning 7 days post-lesion. Tyrosine hydroxylase immunoreactivity (TH-ir) and MB were assessed in the brains of all groups following treatment, and locomotor activity was evaluated prior to lesioning, 7 days post-lesion and after treatment. KEY RESULTS: Increased mitochondrial DNA content and nuclear- and mitochondrial-encoded mRNA and protein expression was observed in specific brain regions of LY344864-treated naïve and lesioned mice, indicating augmented MB. LY344864 attenuated TH-ir loss in the striatum and substantia nigra compared to vehicle-treated lesioned animals. LY344864 treatment also increased locomotor activity in 6-hydroxydopamine lesioned mice, while vehicle treatment had no effect. CONCLUSIONS AND IMPLICATIONS: These data revealed that LY344864-induced MB attenuates dopaminergic neuron loss and improves behavioural endpoints in this model. We suggest that stimulating MB may be beneficial for the treatment of Parkinson's disease and that the 5-HT1F receptor may be an effective therapeutic target.

Mitochondrial-Based Therapeutics for the Treatment of Spinal Cord Injury: Mitochondrial Biogenesis as a Potential Pharmacological Target.

Spinal cord injury (SCI) is characterized by an initial trauma followed by a progressive cascade of damage referred to as secondary injury. A hallmark of secondary injury is vascular disruption leading to vasoconstriction and decreased oxygen delivery, which directly reduces the ability of mitochondria to maintain homeostasis and leads to loss of ATP-dependent cellular functions, calcium overload, excitotoxicity, and oxidative stress, further exacerbating injury. Restoration of mitochondria dysfunction during the acute phases of secondary injury after SCI represents a potentially effective therapeutic strategy. This review discusses the past and present pharmacological options for the treatment of SCI as well as current research on mitochondria-targeted approaches. Increased antioxidant activity, inhibition of the mitochondrial permeability transition, alternate energy sources, and manipulation of mitochondrial morphology are among the strategies under investigation. Unfortunately, many of these tactics address single aspects of mitochondrial dysfunction, ultimately proving largely ineffective. Therefore, this review also examines the unexplored therapeutic efficacy of pharmacological enhancement of mitochondrial biogenesis, which has the potential to more comprehensively improve mitochondrial function after SCI.

Cyclin-Dependent Kinase Inhibitor 1a (p21) Modulates Response to Cocaine and Motivated Behaviors.

This study investigated the functional role of cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) in cocaine-induced responses using a knockout mouse model. Acute locomotor activity after cocaine administration (15 mg/kg, i.p.) was decreased in p21(-/-) mice, whereas cocaine-induced place preference was enhanced. Interestingly, κ-opioid-induced place aversion was also significantly enhanced. Concentration-dependent analysis of locomotor activity in response to cocaine demonstrated a rightward shift in the p21(-/-) mice. Pretreatment with a 5-hydroxytryptamine receptor antagonist did not alter the enhancement of cocaine-induced conditioned place preference in p21(-/-) mice, indicating a lack of involvement of serotonergic signaling in this response. Cocaine exposure increased p21 expression exclusively in the ventral sector of the hippocampus of rodents after either contingent or noncontingent drug administration. Increased p21 expression was accompanied by increased histone acetylation of the p21 promoter region in rats. Finally, increased neurogenesis in the dorsal hippocampus of p21(-/-) mice was also observed. These results show that functional loss of p21 altered the acute locomotor response to cocaine and the conditioned responses to either rewarding or aversive stimuli. Collectively, these findings demonstrate a previously unreported involvement of p21 in modulating responses to cocaine and in motivated behaviors.

Differential effects of cocaine exposure on the abundance of phospholipid species in rat brain and blood.

BACKGROUND: Lipid profiles in the blood are altered in human cocaine users, suggesting that cocaine exposure can induce lipid remodeling. METHODS: Lipid changes in the brain tissues of rats sensitized to cocaine were determined through shotgun lipidomics using electrospray ionization-mass spectrometry (ESI-MS). We also performed pairwise principal component analysis (PCA) to assess cocaine-induced changes in blood lipid profiles. Alterations in the abundance of phospholipid species were correlated with behavioral changes in the magnitude of either the initial response to the drug or locomotor sensitization. RESULTS: Behavioral sensitization altered the relative abundance of several phospholipid species in the hippocampus and cerebellum, measured one week following the final exposure to cocaine. In contrast, relatively few effects on phospholipids in either the dorsal or the ventral striatum were observed. PCA analysis demonstrated that cocaine altered the relative abundance of several glycerophospholipid species as compared to saline-injected controls in blood. Subsequent MS/MS analysis identified some of these lipids as phosphatidylethanolamines, phosphatidylserines and phosphatidylcholines. The relative abundance of some of these phospholipid species were well-correlated (R(2) of 0.7 or higher) with either the initial response to cocaine or locomotor sensitization. CONCLUSION: Taken together, these data demonstrate that a cocaine-induced sensitization assay results in the remodeling of specific phospholipids in rat brain tissue in a region-specific manner and also alters the intensities of certain types of phospholipid species in rat blood. These results further suggest that such changes may serve as biomarkers to assess the neuroadaptations occurring following repeated exposure to cocaine.

ERK oscillation-dependent gene expression patterns and deregulation by stress response.

Studies were undertaken to determine whether extracellular signal regulated kinase (ERK) oscillations regulate a unique subset of genes in human keratinocytes and subsequently whether the p38 stress response inhibits ERK oscillations. A DNA microarray identified many genes that were unique to ERK oscillations, and network reconstruction predicted an important role for the mediator complex subunit 1 (MED1) node in mediating ERK oscillation-dependent gene expression. Increased ERK-dependent phosphorylation of MED1 was observed in oscillating cells compared to nonoscillating counterparts as validation. Treatment of keratinocytes with a p38 inhibitor (SB203580) increased ERK oscillation amplitudes and MED1 and phospho-MED1 protein levels. Bromate is a probable human carcinogen that activates p38. Bromate inhibited ERK oscillations in human keratinocytes and JB6 cells and induced an increase in phospho-p38 and a decrease in phospho-MED1 protein levels. Treatment of normal rat kidney cells and primary salivary gland epithelial cells with bromate decreased phospho-MED1 levels in a reversible fashion upon treatment with p38 inhibitors (SB202190; SB203580). Our results indicate that oscillatory behavior in the ERK pathway alters homeostatic gene regulation patterns and that the cellular response to perturbation may manifest differently in oscillating vs nonoscillating cells.