David M. Thomas

Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines

Neurotoxic amphetamines cause damage to monoamine nerve terminals of the striatum by unknown mechanisms. Microglial activation contributes to the neuronal damage that accompanies injury, disease, and inflammation, but a role for these cells in amphetamine-induced neurotoxicity has received little attention. We show presently that D-methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), D-amphetamine, and p-chloroamphetamine, each of which has been linked to dopamine (DA) or serotonin nerve terminal damage, result in microglial activation in the striatum. The non-neurotoxic amphetamines l-methamphetamine, fenfluramine, and DOI do not have this effect. All drugs that cause microglial activation also increase expression of glial fibrillary acidic protein (GFAP). At a minimum, microglial activation serves as a pharmacologically specific marker for striatal nerve terminal damage resulting only from those amphetamines that exert neurotoxicity. Because microglia are known to produce many of the reactive species (e.g., nitric oxide, superoxide, cytokines) that mediate the neurotoxicity of the amphetamine-class of drugs, their activation could represent an early and essential event in the neurotoxic cascade associated with high-dose amphetamine intoxication.

Tyrosine hydroxylase is inactivated by catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to Parkinson's disease.

Abstract Quinone derivatives of DOPA, dopamine, and N‐acetyldopamine inactivate tyrosine hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl‐catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox‐cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton‐like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox‐active species is highly relevant for neurodegenerative conditions such as Parkinson’s disease.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

MK-801 and dextromethorphan block microglial activation and protect against methamphetamine-induced neurotoxicity

Methamphetamine causes long-term toxicity to dopamine nerve endings of the striatum. Evidence is emerging that microglia can contribute to the neuronal damage associated with disease, injury, or inflammation, but their role in methamphetamine-induced neurotoxicity has received relatively little attention. Lipopolysaccharide (LPS) and the neurotoxic HIV Tat protein, which cause dopamine neuronal toxicity after direct infusion into brain, cause activation of cultured mouse microglial cells as evidenced by increased expression of intracellular cyclooxygenase-2 and elevated secretion of tumor necrosis factor-alpha. MK-801, a non-competitive NMDA receptor antagonist that is known to protect against methamphetamine neurotoxicity, prevents microglial activation by LPS and HIV Tat. Dextromethorphan, an antitussive agent with NMDA receptor blocking properties, also prevents microglial activation. In vivo, MK-801 and dextromethorphan reduce methamphetamine-induced activation of microglia in striatum and they protect dopamine nerve endings against drug-induced nerve terminal damage. The present results indicate that the ability of MK-801 and dextromethorphan to protect against methamphetamine neurotoxicity is related to their common property as blockers of microglial activation.

The newly synthesized pool of dopamine determines the severity of methamphetamine-induced neurotoxicity

The neurotransmitter dopamine (DA) has long been implicated as a participant in the neurotoxicity caused by methamphetamine (METH), yet, its mechanism of action in this regard is not fully understood. Treatment of mice with the tyrosine hydroxylase (TH) inhibitor α‐methyl‐p‐tyrosine (AMPT) lowers striatal cytoplasmic DA content by 55% and completely protects against METH‐induced damage to DA nerve terminals. Reserpine, by disrupting vesicle amine storage, depletes striatal DA by more than 95% and accentuates METH‐induced neurotoxicity. l‐DOPA reverses the protective effect of AMPT against METH and enhances neurotoxicity in animals with intact TH. Inhibition of MAO‐A by clorgyline increases pre‐synaptic DA content and enhances METH striatal neurotoxicity. In all conditions of altered pre‐synaptic DA homeostasis, increases or decreases in METH neurotoxicity paralleled changes in striatal microglial activation. Mice treated with AMPT, l‐DOPA, or clorgyline + METH developed hyperthermia to the same extent as animals treated with METH alone, whereas mice treated with reserpine + METH were hypothermic, suggesting that the effects of alterations in cytoplasmic DA on METH neurotoxicity were not strictly mediated by changes in core body temperature. Taken together, the present data reinforce the notion that METH‐induced release of DA from the newly synthesized pool of transmitter into the extracellular space plays an essential role in drug‐induced striatal neurotoxicity and microglial activation. Subtle alterations in intracellular DA content can lead to significant enhancement of METH neurotoxicity. Our results also suggest that reactants derived from METH‐induced oxidation of released DA may serve as neuronal signals that lead to microglial activation early in the neurotoxic process associated with METH.

Evaluating an intervention for homeless persons: Results of a field experiment

An intensive case management intervention for homeless persons was evaluated by random assignment of 202 cases (involving 213 adults and 70 children) to the intervention or a control group. Full follow-up data (4 interviews: at baseline and at 6-, 12-, and 18-month follow-ups) were available on 98 cases (105 adults and 37 children). The follow-up rates for the 2 groups were not significantly different. Based on 13 repeated measures analyses, there were 3 statistically significant linear time effects (indicating overall change across the follow-up period) and 3 linear Time x Condition interactions (indicating differential change over time for intervention vs. control participants). Regardless of condition, adult participants improved in terms of their experience of homelessness, as well as on physical health symptoms and stressful life events. Condition x Time interactions indicating positive intervention impact were observed on the quality of housing environments, stressful life events, and interviewer ratings of psychopathology.

Dopamine quinones activate microglia and induce a neurotoxic gene expression profile: Relationship to methamphetamine-induced nerve ending damage

Abstract:  Methamphetamine (METH) intoxication leads to persistent damage of dopamine (DA) nerve endings of the striatum. Recently, we and others have suggested that the neurotoxicity associated with METH is mediated by extensive microglial activation. DA itself has been shown to play an obligatory role in METH neurotoxicity, possibly through the formation of quinone species. We show presently that DA‐quinones (DAQ) cause a time‐dependent activation of cultured microglial cells. Microarray analysis of the effects of DAQ on microglial gene expression revealed that 101 genes were significantly changed in expression, with 73 genes increasing and 28 genes decreasing in expression. Among those genes differentially regulated by DAQ were those often associated with neurotoxic conditions including inflammation, cytokines, chemokines, and prostaglandins. In addition, microglial genes associated with a neuronally protective phenotype were among those that were downregulated by DAQ. These results implicate DAQ as one species that could cause early activation of microglial cells in METH intoxication, manifested as an alteration in the expression of a broad biomarker panel of genes. These results also link oxidative stress, chemical alterations in DA to its quinone, and microglial activation as part of a cascade of glial–neuronal crosstalk that can amplify METH‐induced neurotoxicity.

Identification of differentially regulated transcripts in mouse striatum following methamphetamine treatment--an oligonucleotide microarray approach.

Methamphetamine is an addictive drug of abuse that can produce neurotoxic effects in dopamine nerve endings of the striatum. The purpose of this study was to identify new genes that may play a role in the highly complex cascade of events associated with methamphetamine intoxication. Using Affymetrix oligonucleotide arrays, 12 488 genes were simultaneously interrogated and there were 152 whose expression levels were changed following methamphetamine treatment. The genes are grouped into broad functional categories with inflammatory/immune response elements, receptor/signal transduction components and ion channel/transport proteins among the most populated. Many genes within these categories can be linked to ion regulation and apoptosis, both of which have been implicated in methamphetamine toxicity, and numerous factors associated with microglial activation emerged with significant changes in expression. For example, brain‐derived neurotrophic factor (BDNF), chemokine (C‐C) receptor 6 (CCr6) and numerous chemokine transcripts were increased or decreased in expression more than 2.8‐fold. These results point to activated microglia as a potential source of the reactive oxygen/nitrogen species and cytokines that have been previously associated with methamphetamine toxicity and other neurotoxic conditions.

Attenuated microglial activation mediates tolerance to the neurotoxic effects of methamphetamine.

Methamphetamine causes persistent damage to dopamine nerve endings of the striatum. Repeated, intermittent treatment of mice with low doses of methamphetamine leads to the development of tolerance to its neurotoxic effects. The mechanisms underlying tolerance are not understood but clearly involve more than alterations in drug bioavailability or reductions in the hyperthermia caused by methamphetamine. Microglia have been implicated recently as mediators of methamphetamine‐induced neurotoxicity. The purpose of the present studies was to determine if a tolerance regimen of methamphetamine would attenuate the microglial response to a neurotoxic challenge. Mice treated with a low‐dose methamphetamine tolerance regimen showed minor reductions in striatal dopamine content and low levels of microglial activation. When the tolerance regimen preceded a neurotoxic challenge of methamphetamine, the depletion of dopamine normally seen was significantly attenuated. The microglial activation that occurs after a toxic methamphetamine challenge was blunted likewise. Despite the induction of tolerance against drug‐induced toxicity and microglial activation, a neurotoxic challenge with methamphetamine still caused hyperthermia. These results suggest that tolerance to methamphetamine neurotoxicity is associated with attenuated microglial activation and they further dissociate its neurotoxicity from drug‐induced hyperthermia.

Obtaining representative samples of homeless persons: A two-city study

A three-step method for obtaining probability samples of homeless adults across diverse settings over large geographical areas was applied in two cities. Brief surveys (N = 597 in Buffalo + 1,669 in Detroit = 2,266) determined that most homeless people (71–73%) had used a shelter in the prior year and a sizable additional group (18–20%) had used a food program, but not a shelter. Much smaller numbers were found at various in- and out-patient agencies. Less than 1% of the homeless surveyed on the streets used no services in the prior year. These results support the researcher who focuses primarily on shelters and secondarily on food programs in order to obtain a representative sample. A profile of the homeless adult population based on samples receiving full-length interviews (N = 420 + 297 = 717) is provided and compared with prior studies using similarly rigorous probability sampling techniques. Relatively few and generally small differences between the samples from the two cities were found, suggesting that inconsistencies across studies are largely due to methodological rather than geographical factors. Similarly, few and small differences were found among participants obtained across different seasons and sampled from different types of sites (e.g., shelters vs. soup kitchens).© 1999 John Wiley & Sons, Inc.