Jonathan D. Geiger

Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders

Endoplasmic reticulum (ER) is a multifaceted organelle that regulates protein synthesis and trafficking, cellular responses to stress, and intracellular Ca2+ levels. In neurons, it is distributed between the cellular compartments that regulate plasticity and survival, which include axons, dendrites, growth cones and synaptic terminals. Intriguing communication networks between ER, mitochondria and plasma membrane are being revealed that provide mechanisms for the precise regulation of temporal and spatial aspects of Ca2+ signaling. Alterations in Ca2+ homeostasis in ER contribute to neuronal apoptosis and excitotoxicity, and are being linked to the pathogenesis of several different neurodegenerative disorders, including Alzheimers disease and stroke.

Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet

The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect.

Synergistic neurotoxicity by human immunodeficiency virus proteins Tat and gp120: protection by memantine.

Human immunodeficiency virus type 1 (HIV‐1) proteins Tat and gp120 have been implicated in the pathogenesis of dementia associated with HIV infection. Recently, we showed the presence of Tat protein in brains of patients with HIV‐1 encephalitis as well as macaques with encephalitis caused by a chimeric strain of HIV and simian immunodeficiency virus, and that even transient exposure of cells to Tat leads to release of cytopathic cytokines. Now, we report the first demonstration of gp120 protein in brain of patients with HIV encephalitis. We tested the hypothesis that Tat and gp120 would act synergistically to potentiate each proteins neurotoxic effects and determined the extent to which pharmacological antagonists against processes implicated in HIV‐1 neuropathogenesis could block HIV‐1 protein‐induced neurotoxicity. Subtoxic concentrations of Tat and gp120, when incubated together, caused neuronal cell death and prolonged increases in levels of intracellular calcium. A transient exposure of neurons to Tat and gp120 for seconds initiated neuronal cell death, but maximal levels of neuronal cell death were observed with exposures lasting 30 minutes. The neurotoxicity caused by Tat and gp120 applied in combination was blocked completely by memantine, partially by amiloride, and not at all by dipyridamole or vigabatrin. Ann Neurol 2000;47:186–194.

Neurobiological aspects of human immunodeficiency virus infection:Neurotoxic mechanisms

Dementia due to human immunodeficiency virus (HIV) infection is the commonest cause of dementia in children, young adults and middle-aged people. Its incidence continues to rise despite the use of newer antiretroviral agents. The study of the pathogenesis of HIV dementia has lead to the discovery of novel mechanisms of neural dysfunction and toxicity, and holds promise for further discovery of new molecules and pathways involved in maintaining cerebral function and enabling dysfunction. New to the field of neurovirology is the emerging concept that proteins that are part of the virus may themselves be neurotoxic by virtue of their abilities to be either directly toxic to brain cells or may, through their actions on glial cells or macrophages, release neurotoxic products coded by the host genome. These mechanisms have been demonstrated for HIV-1 and accordingly we propose a new term, virotoxins, to describe such actions. This review provides an in-depth analysis of the pathophysiological mechanisms by which these viral and cellular products affected by HIV virotoxins cause neural dysfunction.

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity.

Toxic effects of HIV‐1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV‐1‐infected brain. One such HIV‐1 protein, Tat, can activate calcium release from IP3‐sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate‐ and staurosporine‐mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre‐exposure to Tat. Facilitation of glutamate‐triggered calcium flux by Tat was prevented by inhibitors of ADP‐ribosylation of Gi/Go proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3‐mediated calcium release (xestospongin C), but was not prevented by an activator of Gs (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA‐triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV‐1 associated dementia.

Oxidative stress in HIV demented patients and protection ex vivo with novel antioxidants

Objective: To determine the role of oxidative stress in mediating HIV dementia and to identify novel therapeutic compounds that may block this oxidative stress. Methods: Brain tissue from patients with HIV encephalitis and macaques with simian immune deficiency virus encephalitis was immunostained for lipid peroxidation. Oxidized proteins in CSF of patients with various stages of HIV dementia were quantitated and we determined whether CSF from these patients could alter mitochondrial function. Several novel compounds with antioxidant effects were screened to determine their relative efficacy in protecting against CSF-induced neurotoxicity. Results: Evidence for oxidative stress was present both in brain and in CSF. The presence of oxidized proteins in the CSF and CSF-induced progressive decrease in mitochondrial activity correlated with the severity of cognitive impairment, but only the group of patients with moderate to severe dementia reached statistical significance. l-deprenyl, didox, imidate, diosgenin, and ebselen blocked the CSF-induced toxicity. No effect of trimidox, ruthenium red, or Quercetin was seen. Conclusions: Increased oxidative stress is present in brain and CSF of HIV-infected patients. There is also an accumulation of toxic substances in the CSF that are capable of inducing oxidative stress. The authors have identified several novel compounds that are capable of blocking the CSF-induced toxicity, the therapeutic potential of which is worthy of further exploration.

Involvement of Inositol 1,4,5‐Trisphosphate‐Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV‐1 Protein Tat

Abstract : HIV‐1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS‐related dementia complex. The HIV‐1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat‐induced neurotoxicity. Tat increased [Ca2+]i dose‐dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5‐trisphosphate (IP3)‐mediated intracellular calcium release [8‐(diethylamino)octyl‐3,4,5‐trimethoxybenzoate HCl (TMB‐8) and xestospongin], an inhibitor of receptor‐Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK‐801, (±)‐2‐amino‐5‐phosphonopentanoic acid, and 6,7‐dinitroquinoxaline‐2,3‐dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB‐8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat‐induced neurotoxicity. These results suggest that Tat, via pertussis toxin‐sensitive phospholipase C activity, induces calcium release from IP3‐sensitive intracellular stores, which leads to glutamate receptor‐mediated calcium influx, dysregulation of [Ca2+]i, and Tat‐induced neurotoxicity.

A ketogenic diet suppresses seizures in mice through adenosine A1 receptors

A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.

Neuronal excitatory properties of human immunodeficiency virus type 1 tat protein

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

Adenosine A2A receptor activation reduces proinflammatory events and decreases cell death following intracerebral hemorrhage

The ubiquitous neuromodulator adenosine inhibits the production of several proinflammatory cytokines through activation of specific cell‐surface adenosine receptors. We demonstrated recently that antisense oligonucleotides to tumor necrosis factor‐α (TNF‐α) are neuroprotective in a rat model of intracerebral hemorrhage. Therefore, we hypothesized that activation of adenosine receptors would provide protection against intracerebral hemorrhage‐induced TNF‐α production and inflammatory events. In vitro experiments showed that adenosine A1, A2A, and A3 receptor subtypes were present on U937 cells, and activation of these subtypes inhibited TNF‐α production with a rank order of A2A >> A1 > A3. Prolonged treatment of U937 cells with the A2A receptor agonist 2‐p‐(carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680) desensitized adenosine A2A, A1, and A3 receptors. CGS 21680 administration directly into the striatum immediately prior to the induction of intracerebral hemorrhage inhibited TNF‐α mRNA and, 24 hours following induction, reduced parenchymal neutrophil infiltration (p < 0.001) and TUNEL‐positive cells (p < 0.002) within and bordering the hematoma. These results suggest that pharmacological strategies targeting A2A receptors may provide effective inhibition of acute neurotoxic proinflammatory events that occur following intracerebral hemorrhage.