Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro

Producción CientíficaQuantitation of neurons using stereologic approaches reduces bias and systematic error, but is time-consuming and labor-intensive. Accurate methods for quantifying neurons in vitro are lacking; conventional methodologies are limited in reliability and application. The morphological properties of the soma and neurites are a key aspect of neuronal phenotype and function, but the assays commonly used in such evaluations are beset with several methodological drawbacks. Herein we describe automated techniques to quantify the number and morphology of neurons (or any cell type, e.g., astrocytes) and their processes with high speed and accuracy. Neuronal quantification from brain tissue using a motorized stage system yielded results that were statistically comparable to those generated by stereology. The approach was then adapted for in vitro neuron and neurite outgrowth quantification. To determine the utility of our methods, rotenone was used as a neurotoxicant leading to morphological changes in neurons and cell death, astrocytic activation, and loss of neurites. Importantly, our technique counted about 8 times as many neurons in less than 5-10% of the time taken by manual stereological analysis

Phenothiazine normalizes the NADH/NAD+ ratio, maintains mitochondrial integrity and protects the nigrostriatal dopamine system in a chronic rotenone model of Parkinson's disease

Producción CientíficaImpaired mitochondrial function has been associated with the etiopathogenesis of Parkinson's disease (PD). Sustained inhibition of complex I produces mitochondrial dysfunction, which is related to oxidative injury and nigrostriatal dopamine (DA) neurodegeneration. This study aimed to identify disease-modifying treatments for PD. Unsubstituted phenothiazine (PTZ) is a small and uncharged aromatic imine that readily crosses the blood-brain barrier. PTZ lacks significant DA receptor-binding activity and, in the nanomolar range, exhibits protective effects via its potent free radical scavenging and anti-inflammatory activities. Given that DAergic neurons are highly vulnerable to oxidative damage and inflammation, we hypothesized that administration of PTZ might confer neuroprotection in different experimental models of PD. Our findings showed that PTZ rescues rotenone (ROT) toxicity in primary ventral midbrain neuronal cultures by preserving neuronal integrity and reducing protein thiol oxidation. Long-term treatment with PTZ improved animal weight, survival rate, and behavioral deficits in ROT-lesioned rats. PTZ protected DA content and fiber density in the striatum and DA neurons in the SN against the deleterious effects of ROT. Mitochondrial dysfunction, axonal impairment, oxidative insult, and inflammatory response were attenuated with PTZ therapy. Furthermore, we have provided a new insight into the molecular mechanism underlying the neuroprotective effects of PTZ

Pomegranate juice exacerbates oxidative stress and nigrostriatal degeneration in Parkinson's disease

Producción CientíficaNumerous factors contribute to the death of substantia nigra (SN) dopamine (DA) neurons in Parkinson's disease (PD). Compelling evidence implicates mitochondrial deficiency, oxidative stress, and inflammation as important pathogenic factors in PD. Chronic exposure of rats to rotenone causes a PD-like syndrome, in part by causing oxidative damage and inflammation in substantia nigra. Pomegranate juice (PJ) has the greatest composite antioxidant potency index among beverages, and it has been demonstrated to have protective effects in a transgenic model of Alzheimer's disease. The present study was designed to examine the potential neuroprotective effects of PJ in the rotenone model of PD. Oral administration of PJ did not mitigate or prevent experimental PD but instead increased nigrostriatal terminal depletion, DA neuron loss, the inflammatory response, and caspase activation, thereby heightening neurodegeneration. The mechanisms underlying this effect are uncertain, but the finding that PJ per se enhanced nitrotyrosine, inducible nitric oxide synthase, and activated caspase-3 expression in nigral DA neurons is consistent with its potential pro-oxidant activity

Excitatory amino acids and Alzheimer's disease

Excitatory amino acids (EAA) such as glutamate and aspartate are major transmitters of the cerebral cortex and hippocampus and EAA mechanisms appear to play a role in learning and memory. Anatomical and biochemical evidence suggests that there is both pre- and postsynaptic disruption of EAA pathways in Alzheimer's disease. Dysfunction of EAA pathways could play a role in the clinical manifestations of Alzheimer's disease, such as memory loss and signs of cortical disconnection. Furthermore, EAA might be involved in the pathogenesis of Alzheimer's disease, by virtue of their neurotoxic (excitotoxic) properties. Circumstantial evidence raises the possibility that the EAA system may partially determine the distribution of pathology in Alzheimer's disease and may be important in producing the neurofibrillary tangles, RNA reductions and dendritic changes which characterize this devastating disorder. In this article, we will review the evidence suggesting a role for EAA in the clinical manifestations and pathogenesis of Alzheimer's disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27793/1/0000191.pd

Synaptic localization of striatal NMDA, quisqualate and kainate receptors

Striatal binding of labeled glutamate to (NMDA) receptors, -[alpha]-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) to quisqualate receptors and kainate to kainate receptors was examined in rats which had received unilateral decortications or unilateral striatal quinolinic acid lesions. One week after decortication, there were no significant changes in NMDA, quisqualate or kainate receptors in the striatum ipsilateral to the lesion, when compared to the striatum contralateral to the lesion. In contrast, binding to NMDA receptors was reduced by 92 %, to quisqualate receptors by 80 % and to kainate receptors by 81 % in striatum 3 months after quinolinic acid lesions. The reduction in NMDA receptor binding was significantly greater than the loss of quisqualate or kainate receptors. These results suggest that NMDA, quisqualate and kainate receptor recognition sites are located postsynaptically in the striatum. These results also have implications for the quinolinic acid model of Huntington's disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27879/1/0000293.pd

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Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production

Producción CientíficaIntracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson's disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration

Sodium-dependent -aspartate `binding' is not a measure of presynaptic neuronal uptake sites in an autoradiographic assay

The binding of -[3H]aspartate to sections of rat brain was examined in an autoradiographic assay. Binding was entirely dependent on the presence of sodium ions, but not chloride ions, and was optimal at 2[deg]C. -Aspartate bound rapidly, reached qequilibrium within 20 min and remained stable for 45 min. The rate of dissociation was relatively rapid with a of 56 s, but was not as fast as anticipated, perhaps because of some sequestration of ligand. Binding had a Kd of 6.8 +/- 1.2 [mu]M and a Bmax of 49.4 +/- 8.6 pmol/mg protein. The high Bmax value may further indicate some sequestration of -aspartate. -Glutamate, unlabeled -aspartate, and -threo-hydroxyaspartate, a potent inhibitor of synaptosomal uptake, each competed for -[3H]aspartate binding with IC50s of 7.0 +/- 4.3 [mu]M, 5.4 +/- 1.5 [mu]M, and 2.5 +/- 1.0 [mu]M, respectively. (NMDA), quisqualate, and kainate had no affinity for this site. The regional distribution of -aspartate binding sites was unique and did not conform to the distribution of neuronal uptake sites described by others. Striatal -aspartate binding was unaffected by unilateral decortication or striatal quinolinic acid lesions. In contrast, binding to NMDA, quisqualate, and kainate receptors was reduced by 80-90% by quinolinate lesions of the striatum. The results of -aspartate binding after lesions strongly suggest that this site is not associated with either lesioned glutamatergic afferents or intrinsic neurons of the striatum; it may be associated with glia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28673/1/0000490.pd

A highly reproducible rotenone model of Parkinson's disease

Producción CientíficaThe systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies

Expression of human E46K-mutated α-synuclein in BAC-transgenic rats replicates early-stage Parkinson's disease features and enhances vulnerability to mitochondrial impairment

Producción CientíficaParkinson's disease (PD), the second most common neurodegenerative disorder, is etiologically heterogeneous, with most cases thought to arise from a combination of environmental factors and genetic predisposition; about 10% of cases are caused by single gene mutations. While neurotoxin models replicate many of the key behavioral and neurological features, they often have limited relevance to human exposures. Genetic models replicate known disease-causing mutations, but are mostly unsuccessful in reproducing major features of PD. In this study, we created a BAC (bacterial artificial chromosome) transgenic rat model of PD expressing the E46K mutation of α-synuclein, which is pathogenic in humans. The mutant protein was expressed at levels ~2-3-fold above endogenous α-synuclein levels. At 12 months of age, there was no overt damage to the nigrostriatal dopamine system; however, (i) alterations in striatal neurotransmitter metabolism, (ii) accumulation and aggregation of α-synuclein in nigral dopamine neurons, and (iii) evidence of oxidative stress suggest this model replicates several preclinical features of PD. Further, when these animals were exposed to rotenone, a mitochondrial toxin linked to PD, they showed heightened sensitivity, indicating that α-synuclein expression modulates the vulnerability to mitochondrial impairment. We conclude that these animals are well-suited to examination of gene-environment interactions that are relevant to PD