Christopher A. Shaw

Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death

Oxidative stress has been implicated in both normal aging and in various neurodegenerative disorders and may be a common mechanism underlying various forms of cell death including necrosis, apoptosis, and excitotoxicity. In this review, we develop the hypothesis that oxidative stress-mediated neuronal loss may be initiated by a decline in the antioxidant molecule glutathione (GSH). GSH plays multiple roles in the nervous system including free radical scavenger, redox modulator of ionotropic receptor activity, and possible neurotransmitter. GSH depletion can enhance oxidative stress and may also increase the levels of excitotoxic molecules; both types of action can initiate cell death in distinct neuronal populations. Evidence for a role of oxidative stress and diminished GSH status is presented for Lou Gehrigs disease (ALS), Parkinsons disease, and Alzheimers disease. Potential links to the Guamanian variant of these diseases (ALS-PD complex) are discussed. In context to the above, we provide a GSH-depletion model of neurodegenerative disorders, suggest experimental verifications of this model, and propose potential therapeutic approaches for preventing or halting these diseases.

Glutathione and signal transduction in the mammalian CNS

Abstract : The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its γ‐glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose‐dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)‐2‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Progranulin is expressed within motor neurons and promotes neuronal cell survival.

BackgroundProgranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival.ResultsIn situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival.ConclusionNeurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.

Aluminum in the central nervous system (CNS): toxicity in humans and animals, vaccine adjuvants, and autoimmunity

We have examined the neurotoxicity of aluminum in humans and animals under various conditions, following different routes of administration, and provide an overview of the various associated disease states. The literature demonstrates clearly negative impacts of aluminum on the nervous system across the age span. In adults, aluminum exposure can lead to apparently age-related neurological deficits resembling Alzheimer’s and has been linked to this disease and to the Guamanian variant, ALS–PDC. Similar outcomes have been found in animal models. In addition, injection of aluminum adjuvants in an attempt to model Gulf War syndrome and associated neurological deficits leads to an ALS phenotype in young male mice. In young children, a highly significant correlation exists between the number of pediatric aluminum-adjuvanted vaccines administered and the rate of autism spectrum disorders. Many of the features of aluminum-induced neurotoxicity may arise, in part, from autoimmune reactions, as part of the ASIA syndrome.

Isolation of various forms of sterol β-d-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex

The factors responsible for ALS‐parkinsonism dementia complex (ALS‐PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin‐linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non‐water soluble compounds from washed cycad flour and have identified three sterol β‐d‐glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non‐toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase‐3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS‐PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.

Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour.

Consumption of cycad seed products (Cycas circinalis) is one of the strongest epidemiological links to the Guamian neurological disorder amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), however, the putative toxin which causes neurodegeneration has never been identified definitively. To reexamine this issue, 6–7-mo-old, male CD-1 mice were assessed for motor and cognitive behaviours during and following feeding with pellets made from washed cycad flour. Cycad-fed animals showed early evidence of progressive motor and cognitive dysfunctions. Neurodegeneration measured using TUNEL and caspase-3 labeling was found in neocortex, various hippocampal fields, substantia nigra, olfactory bulb, and spinal cord. In vitro studies using rat neocortex have identified toxic compounds in washed cycad flour that induce depolarizing field potentials and lead to release of lactate dehydrogenase (LDH), both blocked by AP5. High-performance liquid chromatography (HPLC)/mass spectrometry of cycad flour samples failed to show appreciable amounts of other known cycad toxins, cycasin, MAM, or BMAA; only trace amounts of BOAA were present. Isolation procedures employing these techniques identified the most toxic component as β-sitosterol β-d-glucoside (BSSG). The present data suggest that a neurotoxin, or a toxic metabolite, not previously identified in cycad, is able to gain access to central nervous system (CNS) resulting in neurodegeneration of specific neural populations and in motor and cognitive dysfunctions. These data are consistent with a number of major features of ALS-PDC in humans.

Aluminum adjuvant linked to gulf war illness induces motor neuron death in mice

Gulf War illness (GWI) affects a significant percentage of veterans of the 1991 conflict, but its origin remains unknown. Associated with some cases of GWI are increased incidences of amyotrophic lateral sclerosis and other neurological disorders. Whereas many environmental factors have been linked to GWI, the role of the anthrax vaccine has come under increasing scrutiny. Among the vaccine’s potentially toxic components are the adjuvants aluminum hydroxide and squalene. To examine whether these compounds might contribute to neuronal deficits associated with GWI, an animal model for examining the potential neurological impact of aluminum hydroxide, squalene, or aluminum hydroxide combined with squalene was developed. Young, male colony CD-1 mice were injected with the adjuvants at doses equivalent to those given to US military service personnel. All mice were subjected to a battery of motor and cognitive-behavioral tests over a 6-mo period postinjections. Following sacrifice, central nervous system tissues were examined using immunohistochemistry for evidence of inflammation and cell death. Behavioral testing showed motor deficits in the aluminum treatment group that expressed as a progressive decrease in strength measured by the wire-mesh hang test (final deficit at 24 wk; about 50%). Significant cognitive deficits in water-maze learning were observed in the combined aluminum and squalene group (4.3 errors per trial) compared with the controls (0.2 errors per trial) after 20 wk. Apoptotic neurons were identified in aluminum-injected animals that showed significantly increased activated caspase-3 labeling in lumbar spinal cord (255%) and primary motor cortex (192%) compared with the controls. Aluminum-treated groups also showed significant motor neuron loss (35%) and increased numbers of astrocytes (350%) in the lumbar spinal cord. The findings suggest a possible role for the aluminum adjuvant in some neurological features associated with GWI and possibly an additional role for the combination of adjuvants.

THE PLASTICITY-PATHOLOGY CONTINUUM : DEFINING A ROLE FOR THE LTP PHENOMENON

Long‐term potentiation (LTP) is the most widely studied form of neuroplasticity and is believed by many in the field to be the substrate for learning and memory. For this reason, an understanding of the mechanisms underlying LTP is thought to be of fundamental importance to the neurosciences, but a definitive linkage of LTP to learning or memory has not been achieved. Much of the correlational data used to support this claim is ambiguous and controversial, precluding any solid conclusion about the functional relevance of this often artificially induced form of neuroplasticity. In spite of this fact, the belief that LTP is a mechanism subserving learning and/or memory has become so dominant in the field that the investigation of other potential roles or actions of LTP‐like phenomena in the nervous system has been seriously hindered. The multiple subtypes of the phenomena and the myriad molecules apparently involved in these subtypes raise the possibility that observed forms of LTP may represent very different types of modification events, with vastly different consequences for neural function and survival. A relationship between LTP and neuropathology is suggested in part by the fact that many of the molecular processes involved in LTP induction or maintenance are the same as those activated during excitotoxic events in neurons. In addition, some LTP subtypes are clearly induced by pathological stimuli, e.g., anoxic LTP. Such data raise the possibility that LTP is part of a continuum of types of neural modification, some leading to beneficial alterations such as may occur in learning and others that may be primarily pathological in nature, as in kindling and excitotoxicity. In this article, we introduce a plasticity–pathology continuum model that is designed to place the various forms of neural modification into proper context. In vitro and kindling receptor regulation studies are used to provide a basis for evaluating the specific synaptic/cellular response modification along the continuum of events, from beneficial to detrimental, that will be induced by a particular stimulus. J. Neurosci. Res. 58:42–61, 1999.

Lack of behavioral and neuropathological effects of dietary β-methylamino-l-alanine (BMAA) in mice

Beta-methylamino-L-alanine (BMAA) is an excitotoxin allegedly involved in ALS-parkinsonism-dementia complex (ALS-PDC), a neurological disorder found in Guam and its surrounding islands, in which motor neuron disease symptoms can present alone or can co-occur with parkinsonism and dementia. Although in vitro experiments have shown BMAAs neurotoxic properties, studies using adult animals and systemic administration which better model the case of environmentally-induced human neurodegenerative diseases have not supported the involvement of BMAA in these disorders. In order to better test the hypothesized role of BMAA in neurodegeneration, we fed adult mice BMAA at a dose (28 mg/kg body weight, daily for 30 days) that reproduces the natural levels and tested the animals with a battery of behavioural tests, the latter including the evaluation of motor coordination, motor neuron-mediated reflexes, locomotion, muscular strength and memory. We also assessed whether BMAA exposure triggers cell death in the central nervous system (CNS) of mice by examining neuronal numbers and glial response in the spinal cord and the brain. No motor, cognitive or neuropathological outcome resulted from this feeding paradigm. Our findings support neither the causal role of BMAA in neurodegeneration nor the specific involvement of this amino acid in ALS-PDC.

Amyotrophic lateral sclerosis: the involvement of intracellular Ca2+ and protein kinase C

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is characterized by the selective death of motoneurones and corticospinal tract neurones. Abnormalities in excitatory amino acids and their receptors, as well as disordered function of voltage-dependent Ca2+ channels and superoxide dismutase have been reported in ALS patients. Furthermore, the activity of protein kinase C (PKC), a Ca2+, phospholipid-dependent enzyme, is also substantially increased in tissue from ALS patients, suggesting that alterations in intracellular free Ca2+ may be central to many of the diverse pathogenic mechanisms potentially responsible for ALS as discussed here by Charles Krieger and colleagues. Increased PKC activity, in turn, may have direct or indirect effects on neuronal viability and influence the pathogenic process in ALS by modifying the phosphorylation of voltage-dependent Ca2+ channels, neurotransmitter receptors and structural proteins.