Sarah C. Schock

ATP release by way of connexin 36 hemichannels mediates ischemic tolerance in vitro

Spreading depression (SD) is a self-propagating wave of neuronal and glial depolarization that may occur in virtually any gray matter region in the brain. One consequence of SD is an increased tolerance to ischemia. It has been shown that during cortical SD ATP is released into the extracellular space and activation of purinergic receptors leads to the induction of ischemic tolerance. In the present study we show that depolarization of cultured neurons induces ischemic tolerance which is mediated by purinergic receptor activation. Depolarization causes the release of ATP into the extracellular medium, which may be prevented by treatment with the connexin hemichannel blockers flufenamic acid and quinine, but not the pannexin hemichannel blocker carbenoxolone. Knockdown of connexin 36 expression by siRNA greatly reduces the amount of ATP released during depolarization and the subsequent degree of ischemic tolerance. We conclude that during depolarization neurons release ATP by way of connexin 36 hemichannels.

Cortical spreading depression releases ATP into the extracellular space and purinergic receptor activation contributes to the induction of ischemic tolerance

Cortical Spreading Depression (CSD) is a well-studied model of preconditioning that provides a high degree of tolerance to a subsequent ischemic event in the brain. The present study was undertaken in order to determine whether the release of ATP during CSD could contribute to the induction of ischemic tolerance. Direct measurement of ATP levels during CSD indicates that with each CSD wave ATP is released into the extracellular space at levels exceeding 100 microM. Cultures of rat primary cortical neurons exposed to low levels of extracellular ATP developed tolerance to subsequent oxygen-glucose deprivation (OGD) or metabolic hypoxia. The preconditioning effect requires new protein synthesis and develops with time, suggesting that a complex genomic response is required for the induction of tolerance. Multiple purinergic receptors are involved in mediating tolerance, with P2Y receptor activation having the greatest effect. Although extracellular adenosine or glutamate may make a small contribution, most of the tolerance was found to be induced independently of adenosine or glutamate receptor activation. Multiple signal transduction pathways mediate the response to extracellular ATP with the protein kinase A pathway and activation of phospholipase C contributing the most. The results are consistent with the proposal that CSD releases ATP into the extracellular space and the subsequent activation of P2Y receptors makes a major contribution to the induction of ischemic tolerance in the brain.

Rescue of Neurons from Ischemic Injury by Peroxisome Proliferator-Activated Receptor-γ Requires a Novel Essential Cofactor LMO4

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) signaling after stroke may reduce brain injury, but this effect will depend on the levels of receptor and cofactors. Here, we showed that the direct effect of PPARγ signaling to protect neurons from ischemic injury requires a novel cofactor LMO4, because this effect was lost in LMO4-null cortical neurons. PPARγ agonist also failed to reduce cerebral infarction after transient focal ischemia in CaMKIIαCre/LMO4loxP mice with LMO4 ablated in neurons of the forebrain. Expressing LMO4 in LMO4-null cortical neurons rescued the PPARγ-protective effect. PPARγ signaling activates the promoter of the antioxidant gene SOD2 and this process requires LMO4. Addition of a superoxide dismutase mimetic MnTBAP [manganese(III)tetrakis(4-benzoic acid)porphyrin] bypassed the deficiency in PPARγ signaling and was able to directly rescue LMO4-null cortical neurons from ischemic injury. Like LMO4, PPARγ and PGC1α (PPARγ coactivator 1α) levels in neurons are elevated by hypoxic stress, and absence of LMO4 impairs their upregulation. Coimmunoprecipitation and mammalian two-hybrid assays revealed that LMO4 interacts in a ligand-dependent manner with PPARγ. LMO4 augments PPARγ-dependent gene activation, in part, by promoting RXRα (retinoid X receptor-α) binding to PPARγ and by increasing PPARγ binding to its target DNA sequence. Together, our results identify LMO4 as an essential hypoxia-inducible cofactor required for PPARγ signaling in neurons. Thus, upregulation of LMO4 expression after stroke is likely to be an important determinant of neuron survival.

LIM domain only 4 protein promotes granulocyte colony-stimulating factor-induced signaling in neurons

Granulocyte colony-stimulating factor (GCSF) is currently in clinical trials to treat neurodegenerative diseases and stroke. Here, we tested whether LIM domain only 4 protein (LMO4), a hypoxia-inducible gene that protects neurons from ischemic injury, could modulate the neuroprotective effect of GCSF. We showed that GCSF treatment acetylates and phosphorylates Stat3, activates expression of a Stat3-dependent anti-apoptotic gene, p27, and increases neuron survival from ischemic injury. LMO4 participates in Stat3 signaling in hepatocytes and associates with histone deacetylase 2 (HDAC2) in cancer cells. In the absence of LMO4, GCSF fails to rescue neurons from ischemic insults. In wild-type neurons, inhibition of HDAC promoted Stat3 acetylation and the antiapoptotic effect of GCSF. In LMO4 null cortical neurons, expression of wild-type but not HDAC-interaction-deficient LMO4 restored GCSF-induced Stat3 acetylation and p27 expression. Thus, our results indicate that LMO4 enhances GCSF-induced Stat3 signaling in neurons, in part by sequestering HDAC.

Development of dissociated cryopreserved rat cortical neurons in vitro

Dissociated neuronal cultures of various brain regions are commonly used to study physiological and pathophysiological processes in vitro. The data derived from these studies are often viewed to have relevance to processes taking place in the mature brain. However, due to the practical challenges associated with lengthy neuronal culture, neurons are often kept for 14 days in vitro (DIV), or less, before being subject to experimentation. Non-proliferative cultures such as primary neuronal cultures can be maintained for more than 42 DIV if water evaporation from culture media is monitored and corrected. To determine appropriate time points corresponding to the stages of cortical development, we compared characteristics of cryopreserved cortical neurons in cultures at various DIV using immunofluorescence, biochemical measurements and multielectrode array recordings. Compared to 21 and 35 DIV, at 14 DIV, cultures are still undergoing developmental changes and are not representative of adult in vivo brain tissue. Specifically, we noted significant lack in immunoreactivity for synaptic markers such as synapsin, vesicular GABA transporter and vesicular glutamate transporter at 14 DIV, relative to 21 and 35 DIV. Moreover, multielectrode array analysis indicated an increase in network firing up to 46 DIV with patterned firing peaking at 35 DIV. Our results provide specific evidence of the maturational stages of neurons in culture that can be used to more successfully plan various types of in vitro experimentation.

Evaluation of the direct effects of poly(dopamine) on the in vitro response of human osteoblastic cells

Functional polymeric coatings have rapidly become one of the most efficient strategies to endow biomaterials with enhanced bioactive properties. Among the bio-inspired polymers used for biomedical applications, mussel-derived poly(dopamine) (PDA) has increasingly attracted considerable interest because of its unique characteristics. In this work, we carried out detailed physicochemical characterization of a PDA film deposited on nanoporous titanium. In particular, we employed spectroscopic techniques (Raman and ATR-FTIR) and Digital Pulsed Force Mode Atomic Force microscopy (DPFM-AFM) to probe the chemical makeup and the nanomechanical properties of PDA-coated surfaces. In addition, we investigated protein adsorption by ATR-FTIR and quantified it with ten different serum proteins by Liquid Chromatography Mass spectroscopy (LC-MS), aiming at elucidating their potential contribution to the subsequent cell colonization. Successively, we assessed the response of MG-63 human osteoblastic cells to PDA-coated titanium both the multiple- and single-cell levels. Results for this study demonstrate that, compared to bare and nanoporous titanium, the PDA coating positively influences the adhesion and proliferation of MG-63 cells. In addition, we focus on how the three different substrates influence cell morphology (i.e. aspect ratio and form factor), the establishment of focal adhesions and the expression of RhoA, a protein involved in cell contractility. In conclusion, our work provides a deeper insight on the in vitro response of human osteoblastic cells to poly(dopamine) by closing in on specific aspects of cell-PDA interactions, ultimately reaffirming the potential of this bio-inspired polymer as a functional coating for bone tissue engineering applications.

Activation of CRHR1 receptors regulates social and depressive-like behaviors and expression of BDNF and TrkB in mesocorticolimbic regions following global cerebral ischemia

Increased HPA axis activation and CRH release characterize the brains response to global cerebral ischemia. Recently, CRH via activation of CRH type 1 receptors (CRHR1) has been shown to regulate Brain Derived Neurotrophic Factor (BDNF) secretion and emotional behavior. The current study investigates the impact of CRHR1 blockade on BDNF/TrkB signaling expression in the mesolimbic circuitry, and social and depressive-like behavior following global ischemia. Adult male Wistar rats were injected with Antalarmin (2μg/μl) or a vehicle 30min prior to 10min global cerebral ischemia (4VO model) or sham occlusion. The Three Chamber Social Approach Test (SIT) assessed sociability and preference for social novelty, and the novelty suppressed feeding test (NSFT), forced swim test (FST), and sucrose preference test characterized anxiety and depression. Corticosterone levels and organ (thymus, seminal and adrenal glands) weights were determined as additional physiological indices of stress. Immunohistochemistry, Western blot and Rt-PCR were used to assess BDNF and TrkB receptor levels in subregions of the medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and ventral tegmental area (VTA) 30days post-ischemia. Our findings indicate reduced BDNF and TrkB protein and mRNA expression in the mPFC post-ischemia, while heightened levels were found in the NAc. Ischemia increased immobility in the FST and reduced sucrose preference and led to reduced latency to feed in the NSFT and heightened sociability and social novelty preference in the SIT. Antalarmin treatment normalized post-ischemic biochemical/behavioral changes. Our findings support lasting effects of CRHR1 activation on brain plasticity markers, likely playing a role in emotional impairments following cardio- or cerebro-vascular accidents.

The corticostriatal system in dissociated cell culture

The sparse connectivity within the striatum in vivo makes the investigation of individual corticostriatal synapses very difficult. Most studies of the corticostriatal input have been done using electrical stimulation under conditions where it is hard to identify the precise origin of the cortical input. We have employed an in vitro dissociated cell culture system that allows the identification of individual corticostriatal pairs and have been developing methods to study individual neuron inputs to striatal neurons. In mixed corticostriatal cultures, neurons had resting activity similar to the system in vivo. Up/down states were obvious and seemed to encompass the entire culture. Mixed cultures of cortical neurons from transgenic mice expressing green fluorescent protein with striatal neurons from wild-type mice of the same developmental stage allowed visual identification of individual candidate corticostriatal pairs. Recordings were performed between 12 and 37 days in vitro (DIV). To investigate synaptic connections we recorded from 69 corticostriatal pairs of which 44 were connected in one direction and 25 reciprocally. Of these connections 41 were corticostriatal (nine inhibitory) and 53 striatocortical (all inhibitory). The observed excitatory responses were of variable amplitude (−10 to −370 pA, n = 32). We found the connections very secure – with negligible failures on repeated stimulation (approximately 1 Hz) of the cortical neuron. Inhibitory corticostriatal responses were also observed (−13 to −314 pA, n = 9). Possibly due to the mixed type of culture we found an inhibitory striatocortical response (−14 to −598 pA, n = 53). We are now recording from neurons in separate compartments to more closely emulate neuroanatomical conditions but still with the possibility of the easier identification of the connectivity.

Protection conferred by Corticotropin-releasing hormone in rat primary cortical neurons against chemical ischemia involves opioid receptor activation

Different studies have supported neuroprotective effects of Corticotropin-releasing hormone (CRH) against various excitotoxic and oxidative insults in vitro. However, the physiological mechanisms involved in this protection remain largely unknown. The present study was undertaken to determine the impact of CRH administration (at concentrations ranging from 200 fmol to 2 nmol) before and at delayed time intervals following potassium cyanide (KCN)-induced insult in rat primary cortical neurons. A second objective aimed to determine whether kappa and delta opioid receptor (KOR and DOR) blockade, using nor-binaltorphimine and naltrindole respectively (10 microM), could alter CRH-induced cellular protection. Our findings revealed that CRH treatments before or 3 and 8 h following KCN insult conferred significant protection against cortical injury, an effect blocked in cultures treated with alpha-helical CRH (9-41) prior to KCN administration. In addition, KOR and DOR blockade significantly reduced CRH-induced neuronal protection observed 3 but not 8 h post-KCN insult. Using western blotting, we demonstrated increased dynorphin, enkephalin, DOR and KOR protein expression in CRH-treated primary cortical neurons, and immunocytochemistry revealed the presence of opioid peptides and receptors in cortical neurons. These findings suggest protective effects of CRH against KCN-induced neuronal damage, and the contribution of the opioid system in modulating CRH actions.

CRHR1 exacerbates the glial inflammatory response and alters BDNF/TrkB/pCREB signaling in a rat model of global cerebral ischemia: implications for neuroprotection and cognitive recovery

This study examined the impact of corticotropin-releasing hormone type 1 receptor (CRHR1) blockade using Antalarmin (ANT) on the expression of markers of neuroplasticity and inflammation, as well as neuroprotection and behavioral recovery following global cerebral ischemia. Male Wistar rats (N=50) were treated with ANT (2μg/2μl; icv) or a vehicle solution prior to a sham or four vessel (4VO) occlusion. Seven days post ischemia, anxiety was assessed in the Elevated Plus Maze and Open Field tests, and fear and spatial learning in a Y-Maze Passive Avoidance Task and the Barnes Maze. Thirty days post ischemia, brain derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) receptor expression, hippocampal neuronal death and inflammation were determined by analyzing immunoreactivity (ir) of neuron-specific nuclear protein (NeuN), microglia (IBA1, ionized calcium binding adaptor molecule 1), astrocytes (GFAP, glial fibrillary acidic protein) and TNFα (tumor necrosis factor alpha) a pro-inflammatory cytokine. Our findings revealed that ANT improved behavioral impairments, while conferring neuroprotection and blunting neuroinflammation in all hippocampal sub-regions post ischemia. We also observed reduced BDNF and TrkB mRNA and protein levels at the hippocampus, and increased expression at the hypothalamus and amygdala post ischemia, site-specific alterations which were regularized by pre-ischemic CRHR1 blockade. These findings support that CRHR1 actively contributes to altered brain plasticity, neuronal inflammation and injury and recovery of function following ischemic brain insults.