Mitochondrial Toxin 3-nitropropionic Acid Research Articles

Mice with a Partial Deficiency of Manganese Superoxide Dismutase Show Increased Vulnerability to the Mitochondrial Toxins Malonate, 3-Nitropropionic Acid, and MPTP

There is substantial evidence implicating mitochondrial dysfunction and free radical generation as major mechanisms of neuronal death in neurodegenerative diseases. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). In the present study we investigated the susceptibility of mice with a partial deficiency of SOD2 to the neurotoxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3-NP), and malonate, which are commonly used animal models of Parkinson's and Huntington's disease. Heterozygous SOD2 knockout (SOD2+/−) mice showed no evidence of neuropathological or behavioral abnormalities at 2–4 months of age. Compared to littermate wild-type mice, mice with partial SOD2 deficiency showed increased vulnerability to dopamine depletion after systemic MPTP treatment and significantly larger striatal lesions produced by both 3-NP and malonate. SOD2+/− mice also showed an increased production of “hydroxyl” radicals after malonate injection measured with the salicylate hydroxyl radical trapping method. These results provide further evidence that reactive oxygen species play an important role in the neurotoxicity of MPTP, malonate, and 3-NP. These findings show that a subclinical deficiency in a free radical scavenging enzyme may act in concert with environmental toxins to produce selective neurodegeneration.

Beneficial Effects of Dietary Restriction on Cerebral Cortical Synaptic Terminals

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.

Differentially displayed genes in neuroblastoma cells treated with a mitochondrial toxin: evidence for possible involvement of ICAM-1 in 3-nitropropionic acid-mediated neurodegeneration

The mitochondrial toxin 3-nitropropionic acid (3-NPA) causes neurodegeneration in the basal ganglia and neurological symptoms resembling Huntington's disease (HD) when applied to primates or rodents, and therefore might be used as an animal model for this disorder. For that reason, the molecular mechanisms involved in 3-NPA-induced neurodegeneration are of considerable interest. In our model, murine neuroblastoma cells (Neuro-2a) were treated with different doses of 3-NPA, and changes in gene expression were analyzed by means of mRNA differential display (DDRT-PCR). Using 18 primer combinations, we have identified a set of 33 candidate cDNAs deriving from 29 excised DDRT bands whose expression appeared to be changed in response to the 3-NPA insult (mostly elevated). DNA sequencing revealed that novel, as well as previously described genes, are included in this panel. Amongst the known cDNAs, the differential mRNA expression of the ribosomal proteins S6 and L40, of the protein kinase A (PKA) catalytic beta subunit and of the intercellular adhesion molecule ICAM-1 could be verified using Northern hybridization and RT-PCR, respectively. Furthermore, ICAM-1 expression could also be shown to increase at the protein level, which points to a possible function for this molecule in neuronal cells in the course of neurodegeneration. The results may prove useful in elucidating the multiple processes causing neurodegeneration subsequent to lesions by mitochondrial toxins and excitotoxins as well.

Early N-acetylaspartate depletion is a marker of neuronal dysfunction in rats and primates chronically treated with the mitochondrial toxin 3-nitropropionic acid.

N-acetylaspartate (NAA) quantification by 1H-magnetic resonance spectroscopy has been commonly used to assess in vivo neuronal loss in neurodegenerative disorders. Here. the authors used ex vivo and in vivo 1H-magnetic resonance spectroscopy in rat and primate models of progressive striatal degeneration induced by the mitochondrial toxin 3-nitropropionate (3NP) to determine whether early NAA depletions could also be associated with neuronal dysfunction. In rats that were treated for 3 days with 3NP and had motor symptoms, the authors found a significant decrease in NAA concentrations, specifically restricted to the striatum. No cell loss or dying cells were found at this stage in these animals. After 5 days of 3NP treatment, a further decrease in striatal NAA concentrations was observed in association with the occurrence of dying neurons in the dorsolateral striatum. In 3NP-treated primates, a similar striatal-selective and early decrease in NAA concentrations was observed after only a few weeks of neurotoxic treatment, without any sign of ongoing cell death. This early decrease in striatal NAA was partially reversed after 4 weeks of 3NP withdrawal. These results demonstrate that early NAA depletions reflect a reversible state of neuronal dysfunction preceding cell degeneration and suggest that in vivo quantification of NAA 1H-magnetic resonance spectroscopy may become a valuable tool for assessing early neuronal dysfunction and the effects of potential neuroprotective therapies in neurodegenerative disorders.

Major strain differences in response to chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid in rats: implications for neuroprotection studies

Chronic systemic treatment with 3-nitropropionic acid in rats produces persistent dystonia and bradykinesia, and striatal lesions reminiscent of Huntington's disease. However, the interpretation of results obtained with this model are complicated by a heterogeneous distribution of the response to a given toxic dose of 3-nitropropionic acid: approximately half of the animals develop selective striatal lesions, which in certain cases are associated with extrastriatal lesions, and the other half are apparently spared. Thus, the chronic 3-nitropropionic acid lesion model can be difficult for neuroprotection studies in which a consistent response to neurotoxic treatment is prerequisite. We hypothesized that some of the variability in the model was related to the use of Sprague–Dawley rats, since inter-individual variability in response to various stressful conditions has been described previously in this rat strain. We therefore compared 3-nitropropionic acid toxicity in rat strains known to be highly (Fisher 344) or poorly (Lewis) responsive to stress and compared the distribution of responses to that of Sprague–Dawley rats. In a protocol of intraperitoneal injection, toxicity of 3-nitropropionic acid was highest in Fisher rats, intermediate in Sprague–Dawley rats and lowest in Lewis rats. In addition, survival curves showed a more heterogeneous response to 3-nitropropionic acid toxicity in Sprague–Dawley rats than that observed in Lewis and Fisher rats. These differences between Sprague–Dawley and Lewis rats were confirmed in a protocol of subcutaneous 3-nitropropionic acid intoxication using osmotic minipumps, where doses up to 36–45 mg/kg per day for five days were necessary to induce striatal lesions in Lewis rats as compared to 12–14 mg/kg per day for five days in Sprague–Dawley rats. The selectivity of the striatum to lesions, and homogeneous progression of symptoms and neurodegeneration, were more consistently observed in Lewis as compared to Sprague–Dawley rats. These results suggest that vulnerability to 3-nitropropionic acid may depend on genetic factors, which could also influence the physiological response to stress. The present findings also establish an improved model of progressive striatal degeneration in the rat adapted for the testing of new neuroprotective strategies.

Cyclosporin A protects striatal neurons in vitro and in vivo from 3-nitropropionic acid toxicity

The neuroprotective properties of cyclosporin A (CsA) are mediated by its ability to prevent mitochondrial permeability transition during exposure to high levels of calcium or oxidative stress. By using the mitochondrial toxin 3-nitropropionic acid (3NP), the present study assessed whether CsA could protect striatal neurons in vitro and in vivo. In vitro, 3NP produced a 20-30% reduction of striatal glutamic acid decarboxylase-immunoreactive (GAD-ir) neurons. A single treatment with CsA protected GAD-ir neurons from 3NP toxicity at lower (0.2 or 1.0 microM), but not at higher (5.0 microM) doses. Similar findings were seen when the cultures were treated twice with cyclosporin. In vivo experiments used the Lewis rat model of Huntington's disease (HD) in which a low 3NP dose was delivered subcutaneously through an osmotic minipump. Rats received unilateral or bilateral intrastriatal saline injections to disrupt the blood-brain barrier (BBB) and facilitate CsA reaching vulnerable neurons. In the first experiment, CsA treated 3NP-lesioned rats displayed significantly more dopamine-and adenosine-3;, 5;-monophosphate-regulated phosphoprotein (DARPP32-ir) neurons ipsilateral to BBB disruption compared to the contralateral intact striatum, indicating that disruption of the BBB maybe necessary for CsA's neuroprotective effects. In the second experiment, stereological counts of DARPP32-ir neurons revealed that CsA protected striatal neurons in a dose-dependent manner following bilateral disruption of the striatal BBB. Rats treated with the higher (15 or 20 mg/kg) but not lower (5 mg/kg) doses of CsA displayed greater numbers of DARRP32-ir striatal neurons relative to vehicle-treated 3NP-lesioned rats. Thus, under conditions in which CsA can gain access to striatal neurons, significant protection from 3NP toxicity is observed. Therefore, CsA or more lipophilic analogues of this compound, may be of potential therapeutic benefit by protecting vulnerable neurons from the primary pathological event observed in HD.

Neuroprotective role for the p50 subunit of NF-kappaB in an experimental model of Huntington's disease.

Prototypical NF-kappaB consists of a transcription factor dimer of p50 and p65, and an inhibitory subunit called I-kappaB. NF-kappaB is activated in neurons in response to excitotoxic, metabolic, and oxidative stress. Cell-culture data suggest that activation of NF-kappaB can prevent neuronal apoptosis, but its role in vivo is unclear and the specific kappaB subunits involved are unknown. In Huntington's disease (HD), striatal neurons degenerate, and a similar pattern of neuronal vulnerability occurs in rats and mice following exposure to the mitochondrial toxin 3-nitropropionic acid (3NP). We report that mice lacking the p50 subunit of NF-kappaB exhibit increased damage to striatal neurons following administration of 3NP. The neuronal death occurs by apoptosis as indicated by increased caspase activation and DNA fragmentation into oligonucleosomes. NF-kappaB activity is markedly increased in striatum 24-72 h following 3NP administration in wild-type mice, but not in mice lacking p50, indicating that p50 is necessary for the vast majority of 3NP-induced NF-kappaB DNA-binding activity in striatum. Cultured striatal neurons from p50-/- mice exhibited enhanced oxidative stress, perturbed calcium regulation, and increased cell death following exposure to 3NP, suggesting a direct adverse effect of p50 deficiency in striatal neurons.

Rats receiving systemic 3-nitropropionic acid demonstrate impairment of memory in Morris water maze

A recent hypothesis for the etiology of Huntingtons disease (HD) postulates that impaired mitochondrial energy production and/or the presence of reactive free radical species may lead to slow excitotoxic neuronal death. Consistent with this hypothesis, systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) in rats produces selective striatal neuropathology mimicking that seen in HD. Such injections of 3-NP additionally produce motor changes thought to model HD, but possible cognitive changes have not been well described. The present study explores this issue. Sixteen rats underwent acquisition training and subsequent memory assessment in a Morris water maze apparatus. Training over 5 consecutive days consisted of trials during which each rat could escape swimming by finding a permanently located submerged platform. Following training, the rats were divided into two groups and received daily intraperitoneal injections of either 3-NP (15 mg/kg) or saline vehicle for 7 days. On Day 8, a retention trial was conducted, in which the platform was removed and the rats were allowed to swim for 2 min. Swimming patterns were tracked and recorded. The rats receiving 3-NP had impaired memory of the platform location, as represented by decreased time swimming over the platform area, fewer entries into the area, and longer latency to entering the area. These results suggest that the 3-NP rat model produces cognitive dysfunctions that parallel HD dementia.

3-Nitropropionic acid induces a spectrum of Huntington's disease-like neuropathology in rat striatum.

Systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) to rats results in selective striatal lesions and serves as an experimental model of Huntington's disease (HD). However, the effects of the 3-NP treatment are unpredictable and result in lesions of variable severity. The present study was aimed at further characterizing the variability of the striatal lesions induced by systemic administration of 3-NP using osmotic pumps. Hematoxylin-eosin (HE) and Nissl stains as well as immunohistochemical labelling of astrocytes and striatal neurones were performed to analyse the neurotoxic effects of 3-NP. In general, chronic systemic administration of 3-NP resulted in obvious bilateral striatal lesions, which ranged from mild to severe, together with a subtle, but detectable behavioural lesion. Severe type lesions showed marked neuronal loss and an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes surrounding the lesion area, whereas in the core of the lesion GFAP-immunoreactivity was absent. The mild type lesion was characterized by a substantial loss of striatal neurones and an increased expression of GFAP-positive astrocytes throughout the lesion. In a number of 3-NP-treated animals, neither type of lesion was observed, although these animals demonstrated behavioural changes in the paw test compared to controls. In the striatum of these tested 3-NP-treated animals, compromised rk' neurones were detected, suggestive of subtle and early 3-NP-induced neuronal injury. Similar dark neurones were also detected in mild and severe lesions and were immunocytochemically characterized as gamma-aminobutyric acid (GABA) and substance P containing spiny neurones, which belong to the neuronal population that is affected in early HD. These results indicate that systemic administration of 3-NP to rats may result in a spectrum of striatal pathology of which the morphology of the mild type lesion resembles the characteristic HD neuropathology most closely.

Extracellular perfusion of rat brain nuclei using microdialysis: a method for studying differential neurotransmitter release in response to neurotoxins

Microdialysis probes, stereotactically placed in rat brain nuclei, allow detailed kinetic comparisons of neurotransmitter release from in situ chemical lesioning over a continuum from acute early changes (minutes) to chronic late changes (days). This technique insures a localized mechanism of action independent of systemic effects apparent with other routes of administration and independent of mechanical damage patterns encountered in conventional chemical lesioning. The example provided compares changes in extracellular γ-aminobutyric acid (GABA) concentrations in the striatum in response to quinolinic acid (QA, 0.24 M) and 3-nitropropionic acid (3-NPA, 0.25 M). These examples of chemical lesioning represent two theoretical mechanisms of neurodeterioration in Huntington's disease, QA representing the excitotoxic component, and 3-NPA representing the impaired mitochondrial energy component [M.F. Beal, N.W. Kowall, D.W. Ellison, M.F. Mazurek, K.J. Swartz, J.M. Martin, Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid, Nature 321 (1986) 168–171; M.F. Beal, E. Brouillet, B.G. Jenkins, R.J. Ferrante, N.W. Kowall, J.M. Miller, E. Storey, R. Srivastava, B.R. Rosen, B.T. Hyman, Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J. Neurosci. 13 (1993) 4181–4192; N.C. Reynolds, W. Lin, C.M. Cameron, D.L. Roerig, Differential responses of extracellular GABA to intrastriatal perfusions of 3-nitropropionic acid and quinolinic acid in the rat, Brain Res. 778 (1997) 140–149]. An auxillary microdialysis probe implanted in the ipsilateral nucleus accumbens is used to define the physiologic extent of the cytotoxic lesion. Pre-column derivatization of perfusate fractions with o-phthalaldehyde/ t-butylthiol (OPA) provides electroactivity to the OPA-GABA conjugate and facilitates electrochemical detection following high performance liquid chromatography [J.M. Peinado, K.T. McManus, R.B. Myers, Rapid method for microanalysis of endogenous amino acid neurotransmitters in brain perfusates in the rat by isocratic HPLC-EC, J. Neurosci. Methods 18 (1986) 269–276]. Theme: Disorders of the nervous system Topic: Degenerative disease: other

Ablation of the subthalamic nucleus supports the survival of nigral dopaminergic neurons after nigrostriatal lesions induced by the mitochondrial toxin 3-nitropropionic acid

We investigated the role of the excitatory afferents from the subthalamic nucleus (STN) in the death of nigral dopamine (DA) neurons after nigrostriatal axon terminal lesions. Nigral DA neurons were detected by use of both tyrosine hydroxylase immunolabeling or retrograde labeling of nigral cells with fluorogold. Sprague-Dawley rats were subjected to unilateral, quinolinic acid-induced destruction of the STN. Sham lesions of the STN were made by injecting phosphate-buffered saline. Two weeks after STN ablation, lesions of nigrostriatal DA neurons were induced by intrastriatal injections of either the mitochondrial toxin 3-nitropropionic acid (3-NP) or the catecholamine toxin 6-hydroxydopamine (6-OHDA). Intrastriatal injections of 3-NP or 6-OHDA caused a progressive loss of nigral tyrosine hydroxylase-positive or fluorogold-labeled DA neurons in a dose-dependent manner. Previous ablation of the STN significantly attenuates the loss of DA neurons in rats receiving 3-NP but not 6-OHDA. Sham lesions of the STN did not affect DA neuron death induced by the toxins. The results indicate that the excitatory inputs from the STN may contribute to the death of nigral DA neurons under a condition of 3-NP-induced metabolic impairment.

Ablation of the subthalamic nucleus supports the survival of nigral dopaminergic neurons after nigrostriatal lesions induced by the mitochondrial toxin 3‐nitropropionic acid

We investigated the role of the excitatory afferents from the subthalamic nucleus (STN) in the death of nigral dopamine (DA) neurons after nigrostriatal axon terminal lesions. Nigral DA neurons were detected by use of both tyrosine hydroxylase immunolabeling or retrograde labeling of nigral cells with fluorogold. Sprague–Dawley rats were subjected to unilateral, quinolinic acid–induced destruction of the STN. Sham lesions of the STN were made by injecting phosphate-buffered saline. Two weeks after STN ablation, lesions of nigrostriatal DA neurons were induced by intrastriatal injections of either the mitochondrial toxin 3-nitropropionic acid (3-NP) or the catecholamine toxin 6-hydroxydopamine (6-OHDA). Intrastriatal injections of 3-NP or 6-OHDA caused a progressive loss of nigral tyrosine hydroxylase–positive or fluorogold-labeled DA neurons in a dose-dependent manner. Previous ablation of the STN significantly attenuates the loss of DA neurons in rats receiving 3-NP but not 6-OHDA. Sham lesions of the STN did not affect DA neuron death induced by the toxins. The results indicate that the excitatory inputs from the STN may contribute to the death of nigral DA neurons under a condition of 3-NP–induced metabolic impairment. Ann Neurol 1999;45:640–651

Oxidative stress is attenuated in mice overexpressing BCL-2

The protooncogene Bcl-2 inhibits apoptosis in neural cells, which may involve mitochondrial stabilization and decreased generation of reactive oxygen species. Using in vivo microdialysis we found that following administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) there was a significant increase in the conversion of 4-hydroxybenzoic acid (4-HBA) to 3,4-dihydroxybenzoic acid (3,4-DHBA) in control mice, but not in Bcl-2 overexpressing mice. Striatal lesions were observed in littermate control mice, whereas, lesions were minimal or absent in Bcl-2 overexpressing mice. This shows that Bcl-2 overexpression in vivo attenuates the generation of reactive oxygen species.

Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults

Food restriction (FR) in rodents is known to extend life span, reduce the incidence of age-related tumors, and suppress oxidative damage to proteins, lipids, and DNA in several organ systems. Excitotoxicity and mitochondrial impairment are believed to play major roles in the neuronal degeneration and death that occurs in the brains of patients suffering from both acute brain insults such as stroke and seizures, and chronic neurodegenerative conditions such as Alzheimer's, Parkinson's, and Huntington's diseases. We now report that FR (alternate-day feeding regimen for 2-4 months) in adult rats results in resistance of hippocampal neurons to excitotoxin-induced degeneration, and of striatal neurons to degeneration induced by the mitochondrial toxins 3-nitropropionic acid and malonate. FR greatly increased the resistance of rats to kainate-induced deficits in performance in water-maze learning and memory tasks, and to 3-nitropropionic acid-induced impairment of motor function. These findings suggest that FR not only extends life span, but increases resistance of the brain to insults that involve metabolic compromise and excitotoxicity.

Increased vulnerability to 3-nitropropionic acid in an animal model of Huntington's disease.

There is substantial evidence for both metabolic dysfunction and oxidative damage in Huntington's disease (HD). In the present study, we used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of hydroxyl radical production in a transgenic mouse model of HD, as well as in littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was unchanged in the striatum of transgenic HD mice at baseline. Following administration of the mitochondrial toxin 3-nitropropionic acid (3-NP), there were significant increases in 3,4-DHBA generation in both control and transgenic HD mice, and the increases in the transgenic HD mice were significantly greater than those in controls. Furthermore, administration of 3-NP produced significantly larger striatal lesions in transgenic HD mice than in littermate controls. The present results show increased sensitivity to the mitochondrial toxin 3-NP in transgenic HD mice, which suggests metabolic dysfunction in this mouse model of HD.

Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor.

Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 microM) nor dopamine (2.5 microM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 microM) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 microM, reduced the ATP/ADP ratio by approximately 30% after 6 h; this decrease was not augmented by coincubation with 25 microM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.

Glial cell line-derived neurotrophic factor attenuates the locomotor hypofunction and striatonigral neurochemical deficits induced by chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid

The present study investigated whether glial cell line-derived neurotrophic factor prevents the progressive striatal degeneration induced by chronic systemic administration of the mitochondrial toxin, 3-nitropropionic acid. In addition, the effects of delayed treatment with glial cell line-derived neurotrophic factor on toxin-induced behavioural and neurochemical deficits were determined. infused with 3-nitropropionic acid (15 mg/kg/day, for four weeks) via subcutaneous osmotic minipumps was considerably reduced compared to control rats. However, in rats given a single intracerebroventricular injection of 100 μg of glial cell line-derived neurotrophic factor, locomotor activity was significantly higher than in rats injected with the vehicle, an effect that was most pronounced at the onset of toxin infusion. Consistent with a protective or restorative effect in this model of striatal neurodegeneration, toxin-induced deficits in markers of neurotransmitter function were attenuated by glial cell line-derived neurotrophic factor. Thus, [ 3H]GABA uptake and [ 3H]tiagabine/GABA uptake sites in striatal target tissues (globus pallidus and substantia nigra), as well as [ 3H]choline uptake, choline acetyltransferase activity and dopamine receptor binding in the striatum were decreased by the toxin and restored to varying degrees by glial cell line-derived neurotrophic factor administration. As with locomotor abnormalities, effects on neurochemical deficits were most prominent when glial cell line-derived neurotrophic factor was given at the start of toxin infusion, but remained significantly higher than in the vehicle-injected rats when given up to two weeks after. Substance P, dynorphin A and [Met]enkephalin levels in the striatal target tissues also were reduced by 3-nitropropionic acid. The results show that glial cell line-derived neurotrophic factor protects striatal neurons from slow excitotoxic cell death resulting from energy deprivation, secondary to mitochondrial dysfunction. Moreover, they suggest that glial cell line-derived neurotrophic factor may be a viable therapeutic agent for slowly progressive central nervous system disorders, like Huntington's disease, that may be caused by secondary excitotoxicity resulting from abnormal energy utilization.

Mitochondrial toxin 3-nitropropionic acid produces startle reflex abnormalities and striatal damage in rats that model some features of Huntington's disease

Systemic administration of the mitochondrial toxin 3-nitropropionic acid (3NP) to rats produces striatal lesions that mimic some aspects of pathology in Huntington's disease (HD). To evaluate whether 3NP-induced lesions cause sensorimotor gating deficits observed in HD, we measured prepulse inhibition (PPI) of the acoustic startle reflex after systemic administration of 3NP (10, 15, or 20 mg/kg) to 5-month-old rats. PPI, the reduction of startle magnitude by a weak auditory prestimulus, is significantly reduced in patients with HD. Two daily injections of 3NP produced gross histologic evidence of striatal lesions in some rats and significantly reduced PPI. Striatal lesions also significantly disrupted amphetamine-induced stereotypy, another index of dorsal striatal function. 3NP thus reproduces a specific objective and quantifiable gating deficit found in patients with HD.

3-Nitropropionic Acid Neurotoxicity: Visualization by Silver Staining and Implications for Use as an Animal Model of Huntington's Disease

The neuronal damage produced by the mitochondrial toxin 3-nitropropionic acid (3NPA) has been suggested to replicate much of the neuropathology seen in Huntington's disease (HD) and therefore might be used in an animal model. We investigated the susceptibility to this toxin of different neuronal populations in addition to the commonly studied caudate putamen by injecting 3NPA into seven different brain regions as well as systemically. After different survival times, rats were intracardially perfused, brain sections were processed with the Gallyas silver technique, and impregnated neurons were mapped with a computerized microscope. Intracerebral administration of 3NPA resulted in a lesion, the center of which was devoid of tissue while the area was surrounded by a halo of Golgi-like impregnated neurons. In addition to local damage, rats receiving microinjections into the frontal cortex, caudate putamen, basal forebrain, and amygdala displayed argyrophilic neurons in the thalamus corresponding to the topography of thalamofugal neurons projecting to the individual injection sites. On the other hand, negligible secondary damage was seen after injections into the internal capsule, thalamus, or substantia nigra, implicating that thalamofugal axons are especially vulnerable to the local effect of this toxin. Two and a half days after systemic administration of 3NPA, a diffuse argyrophilic neuronal reaction was seen in the dorsolateral part of the caudate putamen, together with a more regionally selective staining of neurons in different cortical areas and the hippocampus. These morphopathological changes were also accompanied by motor deficits. The affected neurons in the cortical regions were primarily in those layers (V and VI) and areas (medial prefrontal, caudal insular/perirhinal, and ventral temporal) that do not project toward the lesioned striatal area; therefore, the cortical pathology may represent another primary site of action of the toxin. Among the affected neurons in the hippocampal complex were pyramidal neurons in the CA1 region as well as various neurons in the CA3 region and dentate hilar area. These studies suggest that a combination of 3NPA administration and a sensitive silver-impregnation method may unravel the potential site of primary neuronal damage in this animal model. Furthermore, these findings may contribute to the understanding of how the disease progresses in HD from the originally affected neuronal population(s) by the recruitment of closely related systems and pathways.

Mechanisms of Cell Death Induced by the Mitochondrial Toxin 3-Nitropropionic Acid: Acute Excitotoxic Necrosis and Delayed Apoptosis

Impaired energy metabolism may play an important role in neuronal cell death after brain ischemia and in late-onset neurodegenerative diseases. Both excitotoxic necrosis and apoptosis have been implicated in cell death induced by metabolic impairment. However, the factors that determine whether cells undergo apoptosis or necrosis are not known. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), a suicide inhibitor of succinate dehydrogenase. Treatment of cultured rat hippocampal neurons with 3-NP resulted in two types of cell death with distinct morphological, pharmacological, and biochemical features. A rapid necrotic cell death, characterized by cell swelling and nuclear shrinkage, could be completely prevented by the NMDA receptor antagonist MK-801 (10 microM) and dose-dependently potentiated by low micromolar levels of extracellular glutamate. A slowly evolving apoptotic death, characterized by nuclear fragmentation, was not attenuated by MK-801 but was prevented by cycloheximide (1 microg/ml). The combination of MK-801 and cycloheximide resulted in an almost complete protection against 3-NP-induced cell death. DNA fragmentation, detected by the terminal deoxynucleotidyl transferase-mediated dUTP-X 3' nick end-labeling technique, was a late event in apoptosis and also occurred after necrotic cell death. ATP depletion was an early event in the 3-NP-induced neuronal degeneration, and the decline in ATP was exacerbated by glutamate. We conclude that 3-NP triggers two separate cell death pathways: an excitotoxic necrosis as a result of NMDA receptor activation and a delayed apoptosis that is NMDA receptor-independent. Mildly elevated levels of extracellular glutamate shift the cell death mechanism from apoptosis to necrosis.