Joseph S. Tauskela

Evaluation of glutathione-sensitive fluorescent dyes in cortical culture

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione‐S‐transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t‐butoxycarbonyl‐Leu‐Met‐7‐amino‐4‐chloromethylcoumarin (CMAC), 7‐amino‐4‐chloromethylcoumarin (CMAC‐blue), monochlorobimane (MCB), and 5‐chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3‐naphthalenedicarboxaldehyde (NDA) and o‐phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL‐buthionine‐(S,R)‐sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC‐blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5–6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC‐blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST‐insensitive OPD also exhibited a decline in GSH‐sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co‐localized with GFAP‐positive astrocytes, but not with MAP‐2‐positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co‐loaded with the Ca2+‐sensitive dye, calcium green 5N‐AM. GLIA 30:329–341, 2000. Published 2000 Wiley‐Liss, Inc.

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-d-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol‐rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen–glucose deprivation (OGD), N‐methyl‐d‐aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only β‐, methylated β‐ and sulfated β‐CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD‐95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X‐100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated β‐CD to protect against NMDA toxicity, and reversed NR2B, PSD‐95 and nNOS localization to Triton X‐100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor‐mediated increases in intracellular Ca2+ concentration ([Ca2+]i), but did suppress OGD‐induced increases in [Ca2+]i. β‐CD, but not Mβ‐CD, also caused a slight block of NMDA‐induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent‐resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol‐rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca2+ levels.

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-d-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

ELEVATED SYNAPTIC ACTIVITY PRECONDITIONS NEURONS AGAINST AN IN VITRO MODEL OF ISCHEMIA

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-d-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the “brink of death.” We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K+-channel blocker, 4-aminopyridine (20–2500 μm), and a GABAA receptor antagonist, bicuculline (50 μm) (4-AP/bic), for 1–2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca2+ spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or l-type voltage-gated Ca2+ channels (L-VGCCs) applied during preconditioning decreased Ca2+ spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca2+/calmodulin-dependent protein kinases (CaMKs) also reduced Ca2+ spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.

Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures.

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen-glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen-glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl-D-aspartate (NMDA) receptor- and Ca(2+)-dependent neurotoxicity underlies oxygen-glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen-glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen-glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen-glucose deprivation preconditioning, underlies oxygen-glucose deprivation tolerance. Several neurotoxic downstream Ca(2+)-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen-glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen-glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen-glucose deprivation or NMDA application. Intracellular Ca(2+) levels, measured using the high- or low-affinity dyes Fluo-4 (K(d) approximately equal 345 nM) or Fluo-4FF (K(d) approximately equal 9.7 microM) respectively, are markedly attenuated during lethal oxygen-glucose deprivation in preconditioned cultures.Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen-glucose deprivation through the suppression of Ca(2+)-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.

Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons.

Excessive μ‐calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non–central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+‐induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 μM) N‐methyl‐D‐aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti‐μ‐calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain‐mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl‐mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non‐CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells. J. Neurosci. Res. 54:223–232, 1998.

An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD‐active MnIIIporphyrins meso‐substituted with N, N′‐dimethylimidazolium or N‐alkylpyridinium groups did not protect neurons against oxygen‐glucose deprivation (OGD), although lower SOD‐active and ‐inactive para isomers protected against N‐methyl‐d‐aspartate (NMDA) exposure. MnIIImeso‐tetrakis(4‐benzoic acid)porphyrin (MnIIITBAP), as well as SOD‐inactive metalloTBAPs and other phenyl ring‐ or β‐substituted metalloporphyrins that contained redox‐insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD‐ or NMDA‐induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor‐mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD‐inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.

Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells.

Cellular volume loss or shrinkage is a ubiquitous feature of apoptosis and thus may contribute to this form of degeneration. Chloride (Cl(-)) and potassium (K(+)) efflux has been shown to participate in volume regulation and several recent reports have implicated K(+) efflux in apoptotic neuronal death. Here pharmacological inhibitors of various K(+) and Cl(-) channels and transporters were used to decipher the relationship between cellular volume regulation and apoptosis. Following exposure to a hypotonic media, cells swell but over time gradually recover, returning to their original cell volume in a process known as regulatory volume decrease (RVD). RVD in N1E 115 neuroblastoma cells was monitored using time-lapse videomicroscopy, cell size and DNA degradation were followed using flow cytometry and fragmented apoptotic nuclei were visualized using Hoechst staining. RVD was blocked by high K(+), TEA and 4-AP (K(+) channel blockers), DIDS and niflumic acid but not SITS (Cl(-) channel blockers), ethacrynic acid (Cl(-) pump blocker), bumetanide (Na(+)/K(+)/Cl(-) cotransporter blocker) and furosemide (K(+)/Cl(-) cotransport blocker). In contrast, only DIDS and SITS (blockers of the Cl(-)/HCO(3) exchanger) inhibited apoptosis, suggesting that a common mechanistic link between RVD and apoptosis is the Cl(-)/HCO(3) exchanger. Thus, this study does not support the notion that K(+) channels are universal anti-apoptotic targets. Instead, the Cl(-)/HCO(3) exchanger may prove to be a viable target of therapeutic intervention for treating pathological apoptosis and neurodegeneration.

Oxygen glucose deprivation-induced astrocyte dysfunction provokes neuronal death through oxidative stress

Understanding the role of astrocytes in stroke is assuming increasing prominence, not only as an important component on its own within the neurovascular unit, but also because astrocytes can influence neuronal outcome. Ischemia may induce astrogliosis and other phenotypic changes, but these remain poorly understood, in part due to limitations in reproducing these changes in vitro. Dibutyryl cyclic AMP-differentiated cultured astrocytes are more representative of the in vivo astroglial cell phenotype, and were much more susceptible than undifferentiated astrocytes to an ischemic-like stress, oxygen-glucose deprivation (OGD). OGD altered the expression/distribution and activity of glial glutamate transporters, impaired cellular glutamate uptake and decreased intracellular levels of glutathione preferentially in differentiated astrocytes. Resistance to OGD was conferred by inhibiting caspase-3 with DEVD-CHO and oxidative stress by the antioxidant N-acetylcysteine (NAC). The resistance of undifferentiated astrocytes to OGD may result from a transient but selective morphological transformation into Alzheimer type II astrocytes, an intermediary stage prior to transforming into reactive astrocytes. Co-culture of neurons with OGD-exposed astrocytes resulted in neurotoxicity, but at surprisingly lower levels with dying differentiated astrocytes. The antioxidant NAC or the 5-LOX inhibitor AA861 added upon co-culture delayed (day 1) but did not prevent neurotoxicity (day 3). Astrocytes undergoing apoptosis as a result of ischemia may represent a transient neuroprotective mechanism via ischemia-induced release of glutathione, but oxidative stress was responsible for neuronal demise when ischemia compromised astrocyte supportive functions.