Emmanuel Brouillet

Age-Dependent Vulnerability of the Striatum to the Mitochondrial Toxin 3-Nitropropionic Acid

Abstract: The mechanisms of delayed onset and cell death in Huntingtons disease (HD) are unknown. One possibility is that a genetic defect in energy metabolism may result in slow excitotoxic neuronal death. Therefore, we examined the effects of age on striatal lesions produced by local administration of the mitochondrial toxin 3‐nitropropionic acid in rats. In vivo chemical shift magnetic resonance imaging showed marked increases in striatal lactate concentrations that significantly correlated with increasing age. Histologic and neurochemical studies showed a striking age dependence of the lesions, with 4‐ and 12‐month‐old animals being much more susceptible than 1‐month‐old animals. Continuous systemic administration of low doses of 3‐nitropropionic acid for 1 month resulted in striatal lesions showing growth‐related changes in dendrites of striatal spiny neurons using the Golgi technique. These results show that a known mitochondrial toxin can produce selective axon‐sparing striatal lesions showing both the age dependence and striatal spiny neuron dendritic changes that characterize HD.

Manganese Injection into the Rat Striatum Produces Excitotoxic Lesions by Impairing Energy Metabolism

There is compelling evidence that excessive exposure to manganese (Mn) produces neurotoxicity, especially in the basal ganglia, resulting in a dystonic Parkinsonian disorder. Several experimental or clinical observations suggest that Mn neurotoxicity could involve impairment of energy metabolism. We examined the neurotoxic effects of Mn following local intrastriatal injection. Three hours after the injection of 2 mumol of MnCl2 into rat striatum, ATP levels were reduced to 51% of the control side and lactate level were increased by 97%, indicating an impairment of oxidative metabolism. Neurochemical analysis of the striata 1 week after Mn injection showed changes consistent with a N-methyl-D-aspartate (NMDA) excitotoxic lesion. Dopamine, gamma-aminobutyric acid, and substance P concentrations showed dose-dependent significant decreases, but concentrations of somatostatin-like immunoreactivity and neuropeptide Y-like immunoreactivity were unchanged. The lesions were blocked by prior removal of the cortico-striatal glutamatergic input or by treatment with the noncompetitive NMDA antagonist MK-801. These findings indicate that Mn neurotoxicity involves a NMDA receptor-mediated process similar to that we have previously found with two characterized mitochondrial toxins, aminooxyacetic acid, and 1-methyl-4-phenylpyridinium. Our results show that Mn may produce neuronal degeneration by an indirect excitotoxic process secondary to its ability to impair oxidative energy metabolism.

1-Methyl-4-Phenylpyridinium Produces Excitotoxic Lesions in Rat Striatum as a Result of Impairment of Oxidative Metabolism

Abstract: The effects of 1‐methyl‐4‐phenylpyridinium (MPP+) were studied in rat striatum. Using freeze‐clamp, microwave, and water‐suppressed proton chemical shift magnetic resonance imaging techniques, MPP+ resulted in marked increases in lactate and a depletion of ATP for up to 48 h after the injections. MPP+ produced dose‐dependent depletions of dopamine, serotonin, γ‐aminobutyric acid, and substance P that were partially blocked at 1 week by prior decortication or completely blocked by MK‐801 at 24 h. The lesions showed relative sparing of somatostatin‐neuropeptide Y neurons, consistent with N‐methyl‐D‐aspartate (NMDA) excitotoxicity. MPP+ produces impairment of oxidative phosphorylation in vivo, which may result in membrane depolarization with persistent activation of NMDA receptors and excitotoxic neuronal degeneration. An impairment of energy metabolism may therefore underlie slow excitotoxic neuronal death in neurodegenerative diseases.

Blockade of 1-methyl-4-phenylpyridinium ion (MPP+) nigral toxicity in the rat by prior decortication or MK-801 treatment: A stereological estimate of neuronal loss

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, (MPTP), produces a parkinsonian syndrome both in man and in experimental animals. Its toxicity is mediated by a metabolite, the 1-methyl-4-phenylpyridinium ion (MPP+). When injected into the striatum, MPP+ is accumulated by dopaminergic nerve terminals and retrogradely transported to the substantia nigra pars compacta (SNc) where it causes neuronal degeneration. MPP+ accumulates in mitochondria and blocks complex I of the electron transport chain. A proposed mechanism of neurotoxicity is excitotoxic neuronal degeneration induced by this energy depletion. We examined whether either prior decortication or administration of the N-methyl-D-aspartate (NMDA) receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) could prevent or diminish the selective nigral neuronal degeneration that follows unilateral intrastriatal injection of MPP+. We quantified the extent of neuronal death in the SNc ipsilateral and contralateral to the injections on Nissl-stained sections with unbiased stereological techniques. One week after injection of MPP+, approximately 75% of the SNc neurons were lost on the side of the injection. The loss was a consequence of the reduction in both SNc volume and neuronal density. Both prior decortication or the administration of MK-801 for 2 days nearly completely prevented MPP(+)-induced neuronal loss in the ipsilateral SNc. These results are consistent with an NMDA receptor mediated excitotoxic mechanism for MPP(+)-induced nigral toxicity.

Non-Invasive Neurochemical Analysis of Focal Excitotoxic Lesions in Models of Neurodegenerative Illness Using Spectroscopic Imaging

Water-suppressed chemical shift magnetic resonance imaging was used to detect neurochemical alterations in vivo in neurotoxin-induced rat models of Huntingtons and Parkinsons disease. The toxins were: N-methyl-4-phenylpyridinium (MPP+), aminooxyacetic acid (AOAA), 3-nitropropionic acid (3-NP), malonate, and azide. Local or systemic injection of these compounds caused secondary excitotoxic lesions by selective inhibition of mitochondrial respiration that gave rise to elevated lactate concentrations in the striatum. In addition, decreased N-acetylaspartate (NAA) concentrations were noted at the lesion site over time. Measurements of lactate washout kinetics demonstrated that t1/2 followed the order: 3-NP ≈ MPP+ » AOAA ≈ malonate, which parallels the expected lifetimes of the neurotoxins based on their mechanisms of action. Further increases in lactate were also caused by intravenous infusion of glucose. At least part of the excitotoxicity is mediated through indirect glutamate pathways because lactate production and lesion size were diminished using unilateral decortectomies (blockade of glutamatergic input) or glutamate antagonists (MK-801). Lesion size and lactate were also diminished by energy repletion with ubiquinone and nicotinamide. Lactate measurements determined by magnetic resonance agreed with biochemical measurements made using freeze clamp techniques. Lesion size as measured with MR, although larger by 30%, agreed well with lesion size determined histologically. These experiments provide evidence for impairment of intracellular energy metabolism leading to indirect excitotoxicity for all the compounds mentioned before and demonstrate the feasibility of small-volume metabolite imaging for in vivo neurochemical analysis.

Systemic or local administration of azide produces striatal lesions by an energy impairment-induced excitotoxic mechanism

Sodium azide is an inhibitor of cytochrome oxidase which produces selective striatal lesions in both rodents and primates. In the present study we investigated the neurochemical and histologic effects of both intrastriatal and systemic administration of sodium azide, as well as the age dependence and mechanism of the lesions. Intrastriatal administration of sodium azide produced dose-dependent lesions. Neurochemical and histologic evaluation showed that markers of both spiny projection neurons (GABA, substance P) and aspiny interneurons (somatostatin, neuropeptide Y, NADPH-diaphorase) were equally affected. Subacute systemic administration of sodium azide resulted in lesions with a similar neurochemical profile; however, in contrast to intrastriatal injections there was sparing of dopaminergic striatal afferents. Prior decortication significantly attenuated lesions produced by intrastriatal administration of sodium azide, consistent with an excitotoxic process. Chronic administration of sodium azide for 1 month lead to striatal neuropathological changes. Lesions produced by intrastriatal administration of sodium azide in 1-, 4-, and 12-month-old animals showed age dependence. Both freeze-clamp measurements and chemical-shift magnetic resonance spectroscopy confirmed that sodium azide impairs oxidative phosphorylation in the striatum following either intrastriatal or systemic administration. These results show that the striatum is particularly vulnerable to oxidative stress produced by sodium azide, and that it produces striatal lesions by a secondary excitotoxic mechanism.

Aminooxyacetic acid striatal lesions attenuated by 1,3-butanediol and coenzyme Q10

We previously showed that intrastriatal administration of aminooxyacetic acid (AOAA) produces striatal lesions by a secondary excitotoxic mechanism associated with impairment of oxidative phosphorylation. In the present study, we show that and the specific complex I inhibitor rotenone produces a similar neurochemical profile in the striatum, consistent with an effect of AOAA on energy metabolism. Lesions produced by AOAA were dose-dependently blocked by MK-801, with complete protection against GABA and substance P depletions at a dose of 3 mg/kg. AOAA lesions were significantly attenuated by pretreatment with either 1,3-butanediol or coenzyme Q10, two compounds which are thought to improve energy metabolism. These results provide further evidence that AOAA produces striatal excitotoxic lesions as a consequence of energy depletion and they suggest therapeutic strategies which may be useful in neurodegenerative diseases.

Systemic Administration of 3-Nitropropionic Acid

Huntington’s disease (HD) is a neurodegenerative disorder characterized by dyskinetic abnormal movements and cognitive decline associated with progressive atrophy of the striatum (1). Generally onset of symptoms occurs in adults and the disease evolves over 10–15 yr toward a fatal outcome. The gene responsible for HD has been identified, and molecular studies of the corresponding encoded protein named huntingtin have made considerable progress (2). However, there are no appropriate phenotypic animal models of the disease based on transgenesis, and the mechanism underlying cell death in HD remains largely unknown.

Induction and Reversal of Cognitive Deficits in a Primate Model of Huntington’s Disease

Huntington’s disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder characterized by involuntary choreiform movements, progressive cognitive decline, psychiatric manifestations, and a neuronal degeneration primarily affecting the striatum. Neuropathological examination indicates that striatal y-amino butyric acid (GABA)ergic projecting neurons are preferentially affected, whereas striatal interneurons are relatively spared (1). The gene responsible for the disease (IT15) has been cloned, and the molecular abnormality has been identified as an expanded polyglutamine tract in the N-terminal region of a protein of unknown function, named huntingtin (2). Recent studies showed that huntingtin interacts with a number of proteins; some of them with well identified functions. Thus, it has been suggested that alterations in glycolysis, vesicle trafficking, or apoptosis may play a role in the physiopathology of HD (3–6). Other data derived from positron emission tomography, magnetic resonance spectroscopy, and postmortem biochemistry, showing evidences for a defect in succinate oxidation, have suggested the potential implication of a primary impairment of mitochondrial energy metabolism (6). Based on this mitochondrial hypothesis, phenotypic animal models of HD have been elaborated both in rodents and nonhuman primates, employing a chronic blockade of succinate oxidation by systemic administration of the mitochondrial toxin, 3-nitropropionic acid (3-NP) (7–11). Historically, initial experimental studies in nonhuman primates used unilateral striatal injections of glutamatergic agonists, such as quinolinic and ibotenic acid, to induce abnormal movements. More recently, experimental studies used a systemic injection of 3-NP in nonhuman primates to induce both choreiform and dystonic movements associated with bilateral selective striatal lesions ressembling those observed in HD (12).