James P. O’Callaghan

Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: Implications for Parkinson's disease

The pathogenic mechanisms underlying idiopathic Parkinsons disease (PD) remain enigmatic. Recent findings suggest that inflammatory processes are associated with several neurodegenerative disorders, including PD. Enhanced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)‐α, has been found in association with glial cells in the substantia nigra of patients with PD. To determine the potential role for TNF‐α in PD, we examined the effects of the 1‐methyl‐4‐phenyl‐1,2,3,4‐tetrahydropyridine (MPTP), a dopaminergic neurotoxin that mimics some of the key features associated with PD, using transgenic mice lacking TNF receptors. Administration of MPTP to wild‐type (+/+) mice resulted in a time‐dependent expression of TNF‐α in striatum, which preceded the loss of dopaminergic markers and reactive gliosis. In contrast, transgenic mice carrying homozygous mutant alleles for both the TNF receptors (TNFR‐DKO), but not the individual receptors, were completely protected against the dopaminergic neurotoxicity of MPTP. The data indicate that the proinflammatory cytokine TNF‐α is an obligatory component of dopaminergic neurodegeneration. Moreover, because TNF‐α is synthesized predominantly by microglia and astrocytes, our findings implicate the participation of glial cells in MPTP‐induced neurotoxicity. Similar mechanisms may underlie the etiopathogenesis of PD.

Work, Obesity, and Occupational Safety and Health

There is increasing evidence that obesity and overweight may be related, in part, to adverse work conditions. In particular, the risk of obesity may increase in high-demand, low-control work environments, and for those who work long hours. In addition, obesity may modify the risk for vibration-induced injury and certain occupational musculoskeletal disorders. We hypothesized that obesity may also be a co-risk factor for the development of occupational asthma and cardiovascular disease that and it may modify the workers response to occupational stress, immune response to chemical exposures, and risk of disease from occupational neurotoxins. We developed 5 conceptual models of the interrelationship of work, obesity, and occupational safety and health and highlighted the ethical, legal, and social issues related to fuller consideration of obesitys role in occupational health and safety.

Divergent Roles for Tumor Necrosis Factor-α in the Brain

Proinflammatory cytokines and chemokines have been implicated in the pathogenesis of several neurological and neurodegenerative disorders. Prominent among such factors is the pleiotropic cytokine, tumor necrosis factor (TNF)-α. Under normal physiological conditions, TNF-α orchestrates a diverse array of functions involved in immune surveillance and defense, cellular homeostasis, and protection against certain neurological insults. However, paradoxical effects of this cytokine have been observed. TNF-α is elicited in the brain following injury (ischemia, trauma), infection (HIV, meningitis), neurodegeneration (Alzheimer’s, Parkinson’s), and chemically induced neurotoxicity. The multifarious identity for this cytokine appears to be influenced by several mechanisms. Among the most prominent are the regulation of TNFα-induced NF-κB activation by adapter proteins such as TRADD and TRAF, and second, the heterogeneity of microglia and their distribution pattern across brain regions. Here, we review the differential role of TNF-α in response to brain injury, with emphasis on neurodegeneration, and discuss the possible mechanisms for such diverse and region-specific effects.

Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: comparison with other methods of sacrifice and analysis of multiple phosphoproteins.

At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.

Phenobarbital and dizocilpine can block methamphetamine-induced neurotoxicity in mice by mechanisms that are independent of thermoregulation

Body temperature profiles observed during methamphetamine (METH) exposure are known to affect dopamine and tyrosine hydroxylase (TH) levels in the striatum of mice; hyperthermia potentiates depletion while hypothermia is protective against depletions. In the current study, the doses of METH were sufficiently great that significant dopamine and TH depletions occurred even though hypothermia occurred. Four doses, administered at 2 h intervals, of 15 mg/kg (4x15 mg/kg) D-METH significantly decreased TH and dopamine levels to 50% of control in mice becoming hypothermic during dosing in a 13 degrees C environment. Phenobarbital or dizocilpine during METH exposure blocked the depletions while diazepam did not. Phenobarbital and dizocilpine did not block depletions by altering the hypothermic profiles from that observed during METH only exposure. Here we show that phenobarbital and dizocilpine can block measures of METH neurotoxicity by non-thermoregulatory mechanisms.

Neurotoxic Effects of Substituted Amphetamines in Rats and Mice

Whether you read about it in the popular press or in the scientific literature, there is no lack of coverage of the issue of amphetamine-induced neurotoxicity. Included among these articles are reports on the adverse effects in both animals and humans of methamphetamine (1–3) and methylenedioxymethamphetamine (MDMA; “Ecstasy”) (4–7). Until it was withdrawn from the market for the potential to affect heart valvular function, the anorectic agent dexfenfluramine drew attention in the experimental and clinical literature for reported neurotoxic effects, even at the prescribed anorectic dosage (8). Lastly and most recently, attention has been focused on the fact that a large percentage of school-age children are maintained on stimulants, including amphetamines, for the treatment of attention deficit/hyperactivity disorder (ADHD) (9). The potential for neurotoxic effects associated with such long-term human exposures is just now being raised (10). Clearly, the term “neurotoxicity” has been very broadly applied to describe the effects of these drugs in both humans and experimental animals. Unfortunately, emphasis has been placed on documenting effects of these agents without distinguishing how and why these effects should be considered “neurotoxic.” Thus, there are many descriptions of drug-induced neurotoxicity but there are very few attempts to link these purported neurotoxic effects to pathological actions on the nervous system or to functional changes meaningful to the human condition. Here, we will briefly review the current status of our understanding of the neurotoxic effects of substituted amphetamines. Emphasis will be placed on defining the neurotoxic condition beyond effects attributable to the neuropharmacological actions of a specific compound. In so doing, we will challenge current dogma with regard to describing neuro-toxic effects of this class of drugs. A detailed and comprehensive review of methamphetamine and amphetamine neurotoxicity recently has appeared (2) and it should be considered the authoritative source on this topic, especially with reference to effects in rats and the modulating role of body temperature. We will cover some of the issues raised by Bowyer and Holson (2), and agree with all of their points, but we will discuss effects of amphetamines in the context of the toxic actions of many known and potential chemical neurotoxicants (11).

Regulatory developmental neurotoxicity testing: a model study focussing on conventional neuropathology endpoints and other perspectives.

Our aim was to investigate a model of the morphologic approach proposed in guidelines for developmental neurotoxicity testing (DNT). Hereto, a limited DNT study [EPA Health Effects Test Guidelines OPPTS 870.6300, 1996a. Developmental Neurotoxicity Study Public Draft, United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA 712-C-96-239, June 1996. ] was carried out with different doses of methylazoxy methanol acetate (MAM), known to affect brain morphology and neuron numbers in the developing brain. After gross examination, the brains of F1-animals were further dissected along neuro-anatomical landmarks to ensure homology between tissues of different individuals. The (relative) weight of the brain (parts) was determined. One brain half (alternating left/right to avoid lateralization) was further used for microscopic slide reading and measurement of brain layer width (linear morphometry); the other was set aside for stereologic investigation in a later phase of the study. In the offspring, a clear reduction in brain size (gross macroscopy) and weight (MAM high- and top-dose groups) was observed on postnatal days (PN) 22 and 62, but this reduction was hard to pinpoint in the microscope as the changes primarily appeared quantitative in nature, rather than qualitative. Linear measurements of brain layer width appeared very sensitive and efficient. This first step of a project is presented and the perspectives of a further stereologic investigation are discussed.

The MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity

ABSTRACT: Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen‐activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth‐associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug‐induced damage to the CNS, we used the dopaminergic neurotoxicant, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme‐linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10‐fold increase in GFAP mRNA by 1 day and a 4‐fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho‐tyr 204 and phospho‐thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady‐state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Immunoblots of these samples showed a number of phosphotyrosine‐labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho‐MAP kinase was elevated over 1.5‐fold, 3–6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury‐induced glial activation.

Health assessment of gasoline and fuel oxygenate vapors: neurotoxicity evaluation.

Sprague–Dawley rats were exposed via inhalation to vapor condensates of either gasoline or gasoline combined with various fuel oxygenates to assess potential neurotoxicity of evaporative emissions. Test articles included vapor condensates prepared from “baseline gasoline” (BGVC), or gasoline combined with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA). Target concentrations were 0, 2000, 10,000 or 20,000 mg/mg3 and exposures were for 6 h/day, 5 days/week for 13 weeks. The functional observation battery (FOB) with the addition of motor activity (MA) testing, hematoxylin and eosin staining of brain tissue sections, and brain regional analysis of glial fibrillary acidic protein (GFAP) were used to assess behavioral changes, traditional neuropathology and astrogliosis, respectively. FOB and MA data for all agents, except G/TBA, were negative. G/TBA behavioral effects resolved during recovery. Neuropathology was negative for all groups. Analyses of GFAP revealed increases in multiple brain regions largely limited to males of the G/EtOH group, findings indicative of minor gliosis, most significantly in the cerebellum. Small changes (both increases and decreases) in GFAP were observed for other test agents but effects were not consistent across sex, brain region or exposure concentration.

Prior exposure to corticosterone markedly enhances and prolongs the neuroinflammatory response to systemic challenge with LPS

Systemic exposure to the inflammagen and bacterial endotoxin lipopolysaccharide (LPS) has been widely used to evaluate inflammation and sickness behavior. While many inflammatory conditions occur in the periphery, it is well established that peripheral inflammation can affect the brain. Neuroinflammation, the elaboration of proinflammatory mediators in the CNS, commonly is associated with behavioral symptoms (e.g., lethargy, anhedonia, anorexia, depression, etc.) termed sickness behavior. Stressors have been shown to interact with and alter neuroinflammatory responses and associated behaviors. Here, we examined the effects of the stress hormone, corticosterone (CORT), as a stressor mimic, on neuroinflammation induced with a single injection (2mg/kg, s.c.) or inhalation exposure (7.5 μg/m3) of LPS or polyinosinic:polycytidylic acid (PIC; 12mg/kg, i.p.) in adult male C57BL/6J mice. CORT was given in the drinking water (200 mg/L) for 1 week or every other week for 90 days followed by LPS. Proinflammatory cytokine expression (TNFα, IL-6, CCL2, IL-1β, LIF, and OSM) was measured by qPCR. The activation of the neuroinflammation downstream signaling activator, STAT3, was assessed by immunoblot of pSTAT3Tyr705. The presence of astrogliosis was assessed by immunoassay of GFAP. Acute exposure to LPS caused brain-wide neuroinflammation without producing astrogliosis; exposure to CORT for 1 week caused marked exacerbation of the LPS-induced neuroinflammation. This neuroinflammatory “priming” by CORT was so pronounced that sub-neuroinflammatory exposures by inhalation instigated neuroinflammation when paired with prior CORT exposure. This effect also was extended to another common inflammagen, PIC (a viral mimic). Furthermore, a single week of CORT exposure maintained the potential for priming for 30 days, while intermittent exposure to CORT for up to 90 days synergistically primed the LPS-induced neuroinflammatory response. These findings highlight the possibility for an isolated inflammatory event to be exacerbated by a temporally distant stressful stimulus and demonstrates the potential for recurrent stress to greatly aggravate chronic inflammatory disorders.