Selective Neurotoxicity Research Articles

Selective neurotoxicity of ruthenium red in primary cultures

The inorganic dye ruthenium red (RuR) has been shown to be neurotoxic in vivo when injected intracerebrally. In this work the toxicity of RuR was compared in primary cultures of rat cortical neurons, cerebellar granule neurons and cerebellar astroglia. Microscopic examination of the cultures revealed that RuR penetrates the somata of both types of neurons used and produces vacuolization and loss and fragmentation of neurites. In contrast, no RuR was seen inside cultured astrocytes and no morphological signs of damage were observed in these cells. RuR toxicity was also assessed by immunocytochemistry of alpha-tubulin and by biochemical measurement of the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the cultured cells. The morphological alterations in the neurons were closely correlated with loss of tubulin immunoreactivity and particularly with a notable decrement in the ability to reduce MTT. Using the latter parameter, it was found that neuronal damage was independent of the age of the cultures, augmented progressively with time of incubation with RuR, from 8 to 24 h, and showed a clear dose-response curve from 20 to 100 microM RuR. Astrocytes showed only a slight decrease in MTT reduction after 24 h of incubation with 100 microM RuR. It is concluded that RuR seems to be toxic for neurons but not for astroglia, and that this selectivity is probably related to the ability of the neurons to internalize the dye. The possible mechanisms of RuR penetration and neuronal damage are discussed.

Serotonin and alcohol-related brain damage.

Preliminary results from the immunohistochemical examination of the brainstems of chronic alcoholics, suggest that alcohol may have a role in damage to the principal serotonergic (5HT) nuclei. This view is reinforced by evidence from previous animal experiments which demonstrated a reduction in 5HT neurons in the brains of alcohol-preferring rats and selective neurotoxicity to 5HT neurons following 5,6-dihydroxytryptamine-induced increased ethanol intake. It is speculated that, like other neurotoxins, alcohol or its metabolites cause degeneration of 5HT axons and axon terminals. It is possible that if axonal damage is sufficiently severe and chronic, the eventual consequence is cell death. This could be due to insufficient opportunity for repair and regrowth under repeated and sustained insults of high alcohol consumption.

Axonal transport of manganese and its relevance to selective neurotoxicity in the rat basal ganglia

The present study provides evidence for anterograde axonal transport of manganese (Mn) in the basal ganglia. Microinjections of 54Mn into rat substantia nigra or striatum revealed region-specific accumulation and retention of the isotope in globus pallidus, striatum, thalamus and substantia nigra for up to at least 48 or 72 h respectively. Within 4 h after intrastriatal injection of 54Mn, radioactivity accumulated in the substantia nigra, suggesting axonal transport of the metal. Subsequent studies using bilateral 54Mn injections into striatum or substantia nigra and unilateral colchicine injections into or transection of the medial forebrain bundle confirmed axonal transport of Mn through these fibres. Selective destruction of the striatonigral or nigrostriatal pathways using quinolinic acid or 6-hydroxydopamine 2 weeks before injection of the isotope, revealed uptake of 54Mn by cell bodies of both γ-aminobutyric acidergic striatal and dopaminergic nigral neurons and subsequent anterograde transport through sriatonigral or nigrostriatal fibres. In addition, the quinolinic acid-lesioned striatum three times more radioactivity than the intact striatum. In conclusion, the present data suggest that both glial cells and striatonigral and nigrostriatal neurons are potential targets for Mn toxicity. These results and the selective neurotoxicity of Mn are discussed with respect to the iron transport protein transferrin, transferrin receptors, the iron storage protein ferritin, and mitochondrial dysfunction.

Late-onset selective neuronal damage in the rat spinal cord induced by continuous intrathecal administration of AMPA

Neurotoxicity mediated by 1-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was investigated by infusing this agent continuously for 7 days intrathecally to adult rats using a mini-osmotic pump. Behavioral changes were apparent only after the second postoperative day, when the rats displayed hindlimb palsy or incontinence of urine. The behavioral deficits became progressively severe and the rats usually displayed both hindlimb paraplegia and incontinence of urine by the 7th postoperative day. These progressive behavioral deficits were induced in a dose-dependent manner in the rats that received AMPA at a dose of 100pmol/h (100 μM at 1 μ1/h, 17 nmol in total dose). The severity of behavioral deficits was in parallel with that of neuropathological changes in the lumbosacral cords. In spinal segments rostrally adjacent to those with severe pathological changes, only the neurons in the dorsal horns (Rexed's laminae II–IV) were destroyed with intense gliosis. These changes were not induced by infusing AMPA for 1 day. The concomitant administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist for non- N-methyl- d-aspartate (NMDA) receptors, with AMPA, but not that of 2-amino-5-phosphonovalerate (APV), an antagonist for NMDA receptor, prevented induction of the behavioral and neuropathological changes. The findings of the present study suggest that this late-onset, selective neurotoxicity is mediated by AMPA-type glutamate receptors.

N-methyl-4-phenylpyridinium (MPP +) potentiates the killing of cultured hepatocytes by catecholamines

The role of catecholamines in the toxicity of MPTP ( N-methyl-4-pheny-1,2,3,6-tetrahydropyridine) was explored. The killing of cultured hepatocytes by dopamine and 6-hydroxydopamine was enhanced following inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a manipulation known to sensitize such cells to an oxidative stress. The participation of activated oxygen species in the cell injury under such circumstances was shown by the ability of catalase and the ferric iron chelator deferoxamine to protect the hepatocytes. The toxicity of catecholamines was also potentiated by the mitochondrial site I (NADH dehydrogenase) inhibitor rotenone. MPP + ( N-methyl-4-phenylpyridinium), the putative toxic metabolite of MPTP is also a site I inhibitor. Incubation of hepatocytes with MPP + similarly potentiated the toxicity of 6-hydroxydopamine, dopamine, and norepinephrine under conditions where MPP + alone or catecholamines alone did not kill cells. Hepatocytes that had accumulated dopamine from the medium were killed by a subsequent exposure to MPP + in the absence of a catecholamine in the medium. Hepatocytes that had not been pretreated with dopamine were not affected by the subsequent exposure to MPP +. These data indicated that catecholamines render hepatocytes more susceptible to the toxicity of MPP + and suggest that the presence of catecholamines in specific neurons in the brain may be related to the selective neurotoxicity of MPTP.

Selective neurotoxicity induced by lasalocid in dissociated cerebral cultures

Dispersed cerebral cells prepared from 15–16-day mouse foetuses were cultured for 7 days and exposed to lasalocid for periods of 4, 18 or 48 hr. Cultures were examined by phase contrast microscopy and processed for scanning electron microscopy. Lasalocid (1 and 2 μ m) induced neurotoxic effects that were evident already at 4 hr, including swelling of perikarya, followed by cytolysis of most neurons present in the cultures. During the following 18 hr virtually the entire neuronal population degenerated completely. Cultures exposed to a lower concentration of lasalocid (0.5 μ m) for 48 hr showed only mild damage to some neurons, and at 0.2 μ m no damage could be observed, even after several days of continuous exposure. Notably, glial and other non-neuronal cells were not damaged by exposure to 2 μ m-lasalocid, and cell division resumed on returning the cultures to regular growth media. Similarly, lasalocid (2 μ m) was not toxic to cultured rat astrocytes. These morphological observations were followed by measurements of 45Ca 2+ influx in the dissociated cerebral cultures. Lasalocid (1 μ m) induced 45Ca 2+ influx of 40% above dimethyl sulphoxide control values. The voltage-sensitive calcium channel antagonists nimodipine (10 μ m) and D-600 (50 μ m) did not inhibit lasalocid-mediated 45Ca 2+ influx, whereas MK-801 [10 μ m; a non-competitive N-methyl- d-aspartate (NMDA) receptor/channel antagonist] exclusively blocked this influx and prevented cytotoxic damage. Conversely, NMDA and glutamate, which by themselves mediated 45Ca 2+ influx in these cultures, both potentiated lasalocid-induced 45Ca 2+ influx and cellular damage. These observations clearly demonstrate the selective neurotoxicity of lasalocid to cultured cerebral neurons, and may imply involvement of the NMDA receptor/channel in lasalocid-mediated neurotoxicity.

Selective neurotoxicity of COOH-terminal fragments of the β-amyloid precursor protein

The primary component of amyloid deposits found in the brains of patients with Alzheimer's disease is the β-amyloid protein, a derivative of a much larger precursor protein (βPP). We have previously reported that overexpression of carboxyl (COOH)-terminal fragments of βPP from an integrated DNA construct leads to degeneration of neuronally differentiating mouse embryonic stem cells and that the neuronal degeneration is related to approximately 14- and 15-kDa COOH-terminal fragments of the precursor protein. We here demonstrate that these putative cytotoxic fragments contain intact β-amyloid protein. When such transformed cell lines are treated with dimethyl sulfoxide to induce differentiation into muscle cells, however, the resulting muscle cells remain viable (as do control non-transformed cells), despite the production of comparable amounts of the 14- and 15-kDa fragments. These results are consistent with the hypothesis that particular COOH-terminal fragments of βPP are amyloidogenic and neurotoxic.

Further insights into the molecular mechanisms of action of the serotonergic neurotoxin 5,7-dihydroxytryptamine.

Autoxidation and various enzyme-mediated oxidations of the serotonergic neurotoxin 5,7-dihydroxytryptamine (1) give 5-hydroxytryptamine-4,7-dione (2) and 6,6'-bi(5-hydroxytryptamine-4,7-dione) (3) as the major products. When administered into the ventricular system of mice 2 and 3 are general toxins. The LD50 values for 2 (29.6 +/- 0.04 micrograms) and 3 (25.4 +/- 0.30 micrograms) are lower than that for 1 (51.8 +/- 0.28 micrograms). In the presence of cellular reductants (glutathione, cysteine, ascorbate) and molecular oxygen, or when incubated with rat brain homogenate, 2 and 3 redox cycle and form superoxide radical anion, O2.-, as a byproduct. The lethal effects of 2 and 3 when introduced into the brain may in part be due to such redox cycling reactions which deplete oxygen levels and, as a result of Haber-Weiss chemistry deriving from O2.-, form the cytotoxic hydroxyl radical (HO.). Intraventricular administration of 2 and 3 to mice causes only relatively minor and transient (ca. 1 h to 1 day) changes in whole brain levels of dopamine, 5-hydroxytryptamine (from both 2 and 3), acetylcholine, and choline (from 2 only). These changes differ from the profound and long-lasting serotonergic deficit evoked by 1. On the basis of these results a hypothesis has been formulated which proposes that the selective neurotoxicity of 1 derives from its rapid uptake into serotonergic neurons where it is oxidatively converted to 2 and 3. Redox cycling reactions of 2 and 3 then result in the depletion of intraneuronal oxygen and concomitant formation of O2.-. Dismutation of O2.- gives H2O2 which, as a result of transition metal ion-catalyzed Haber-Weiss chemistry, yields HO.. Thus, neuronal damage and death might result from the combined effects of hypoxia and HO. formation.

Expression of a carboxy-terminal region of the β-amyloid precursor protein in a heterogeneous culture of neuroblastoma cells: evidence for altered processing and selective neurotoxicity

Six independent clonal isolates from a morphologically heterogeneous human neuroblastoma cell line stably expressed several products of the human amyloid precursor protein (APP) from an introduced DNA construct; the “substrate-adherent” phenotype (fibroblast-like cells) predominated in all 6; these displayed immunoreactivity of vimentin, but little to no reactivity of neuron-specific enolase. A stably transfected isolate which did not show any expression from the identical construct (presumably because of a position effect) exhibited the predominantly neuronal phenotype of the parental cells (neuron-specific enolase positive). These results suggest selective neurotoxicity of the expressed products. Two of the 6 stably expressing cell lines showed a decrease of native mRNA for APP to levels that were 1 4 - 1 3 that of the parental cells and a decrease of their growth rates to half that of the parental cells; these decreased growth rates were improved by conditioned medium from the parental cell line. Western blot analysis revealed at least four distinct fragments of the COOH-terminus of APP in the isolate which expressed protein and mRNA in greatest abundance, suggesting that overexpression of APP in a human neural cell line leads to aberrant cleavage of APP.

Alcohol as a social teratogen

This chapter reviews the effect of alcohol on the behavioral and neurochemical aspects of mammalian development. Evidence is presented in the chapter to suggest that the behavioral changes seen in the rat following pre-natal exposure to the drug may bear some similarity to those seen in children suffering from the foetal alcohol syndrome. The term foetal alcohol effects have recently been suggested by Streissguth and Martin to describe the less extreme changes. In common with other birth defects, affected children show considerable individual variation regarding the severity of the damage. Despite the wide recognition of the existence of the foetal alcohol syndrome, controversy exists regarding the importance of alcohol exposure as the sole factor causing the syndrome. The effects of alcohol on the lipid composition of the neuronal membranes, and on the function of neural cell adhesion molecules (N-CAM) during critical stages of neuro-ontogenesis, are probably of primary importance in determining the selective neurotoxicity of alcohol.

Suppression of MPTP-induced dopaminergic neurotoxicity in mice by nomifensine and l-DOPA

To examine effects of various pharmacological manipulations of dopamine (DA) metabolism on DA neurotoxicity of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), C57 black mice were injected with MPTP (30 mg/kg s.c., once daily for two days) alone or in combination with apomorphine, bromocriptine, haloperidol, l-DOPA or nomifensine. MPTP markedly decreased neostriatal DA concentrations at 2, 10, 20 and 30 days post-treatment indicating persistent degeneration of nigrostriatal DA neurons. Suppression or acceleration of DA turnover rates by the DA agonists apomorphine and bromocriptine or by the DA antagonist haloperidol, respectively, did not affect MPTP toxicity. MPTP-induced neostriatal DA depletions were markedly suppressed by nomifensine, a DA reuptake inhibitor, and attenuated by exogenous l-DOPA. MPTP may be a substrate for the DA reuptake system and its specific transport into nigrostriatal terminals may be an important factor for its selective neurotoxicity.

IV. Neurotoxicity of colchicine and other tubulin-binding agents: A selective vulnerability of certain neurons to the disruption of microtubules

Colchicine and certain other agents which disrupt microtubules and interfere with axonal and dendritic transport are highly toxic to certain CNS neurons. The present chapter summarizes our knowledge about this selective neurotoxicity. Injections of colchicine into several brain regions lead to the death of selected populations of neurons within those regions. Intra-hippocampal injections selectively destroy granule cells of the dentate gyrus; hippocampal pyramidal cells are essentially unaffected. Injections into the cerebellum, olfactory bulb, and caudate nucleus also destroy resident neurons. In these areas several cell types are vulnerable. Neurons of the cerebral cortex appear to be much less affected by colchicine, although some neurons of paleocortical regions are vulnerable. Colchicine does not appear to be an excitotoxin like kainic acid. The neurotoxicity of colchicine appears to be related to the destruction of microtubules, since other agents which disrupt microtubules have similar toxic effects, and since analogs of colchicine which do not disrupt microtubules are non-toxic. Colchicine may induce an autotoxic response which leads to neuronal death in certain populations due to the accumulation of some toxic cellular product which is normally transported by a microtubule-dependent process. The selective vulnerability of neurons to the neurotoxic effects of colchicine may be a model for system degenerations of the central nervous system in which certain subpopulations of neurons are selectively vulnerable to abnormal accumulations of metabolic products.

Intracellular transport in neurons.

Intracellular transport in neurons.

Cholinesterase in the chicken nervous system.

WHILE making a topographical survey of the distribution of cholinesterases in the spinal cord and brain stem of the chicken in relation to the question of the selective neurotoxicity of certain organophosphorus compounds, we were led to make a comparison in four laboratory mammals (to be published). Koelle1 had shown histochemically in the rat that considerable variation exists in the cholinesterase content of neurones from one centre to another. Hebb2 also recognized in mammals neurones where the cholinesterase was for the most part intracellular (cholinergic) and neurones where it was largely distributed on the neuronal membrane (cholinoceptive). In contrast to this variability in mammals, the distribution of cholinesterase in chicken neurones is strikingly uniform.