Protein nitration in Parkinson's disease.

Oxidative stress has been proposed as a pathogenetic mechanism in Parkinson's disease (PD). One mechanism of oxidative cellular injury is the nitration of protein tyrosine residues, mediated by peroxynitrite, a reaction product of nitric oxide and superoxide radicals. We demonstrate here the presence of nitrotyrosine immunoreactivity in Lewy bodies within melanized neurons and in amorphous deposits associated with intact and degenerating neurons. The core of the Lewy body was frequently intensely immunolabeled, while the rim was lightly labeled or unlabeled. This likely reflects the fact that tyrosine residues of neurofilament proteins are primarily localized to Lewy body cores, and suggests that nitrotyrosine is present in neurofilament protein itself. Although these observations are as yet unable to provide a definitive link between oxidative stress and neuronal dysfunction, they demonstrate that oxidative stress has occurred within the vulnerable neurons of PD, leaving a permanent marker of oxidative modification of neuronal proteins within the target cells of neurodegeneration. In addition, these observations provide a potential link between excitotoxicity and oxidative stress within the vulnerable neurons of PD and represent a pathogenetic mechanism in common with the 2 other major age-related neurodegenerative diseases, Alzheimer disease and amyotrophic lateral sclerosis.

Cholinergic function in lumbar aluminum myelopathy.

To determine whether perikaryal neurofilamentous accumulation in cholinergic neurons is associated with a deficit in cholinergic function, we developed a new model of aluminum-induced neurofibrillary degeneration, referred to as focal lumbar aluminum myelopathy. The model is produced by direct intramedullary microinjection of AlCl3, which results in a characteristic neurological syndrome. Four weeks after injections, affected rabbits show extensive neurofilamentous lesions of both large and small neurons in the lumbar spinal cord, including a majority of anterior horn cells. These animals are capable of long-term survival. Posterior tibial nerve morphometry in these rabbits revealed no significant loss of myelinated fibers. Choline acetyltransferase (ChAT) activity in the sciatic nerve was decreased 39%, from 45.70 +/- 2.36 nmol ACh/hour/3-mm segment in acid-injected controls to 17.72 +/- 1.94 in aluminum-intoxicated rabbits. The rate of accumulation of ChAT activity proximal to a sciatic nerve ligature was significantly greater in the aluminum-treated rabbits, although the total amount of ChAT activity accumulating in a 24-hour period did not differ from controls. We conclude that aluminum-induced accumulation of neurofilaments in cholinergic perikarya is associated with a sharp decrease of ChAT activity in the axons of those cells and possibly with a compensatory increase in the rate of delivery of the enzyme.

Morphology and kainate-receptor immunoreactivity of identified neurons within the entorhinal cortex projecting to superior temporal sulcus in the cynomolgus monkey.

Projections of the entorhinal cortex to the hippocampus are well known from the classical studies of Cajal (Ramon y Cajal, 1904) and Lorente de Nó (1933). Projections from the entorhinal cortex to neocortical areas are less well understood. Such connectivity is likely to underlie the consolidation of long-term declarative memory in neocortical sites. In the present study, a projection arising in layer V of the entorhinal cortex and terminating in a polymodal association area of the superior temporal gyrus has been identified with the use of retrograde tracing. The dendritic arbors of neurons giving rise to this projection were further investigated by cell filling and confocal microscopy with computer reconstruction. This analysis demonstrated that the dendritic arbor of identified projection neurons was largely confined to layer V, with the exception of a solitary, simple apical dendrite occasionally ascending to superficial laminae but often confined to the lamina dissecans (layer IV). Finally, immunoreactivity for glutamate-receptor subunit proteins GluR 5/6/7 of the dendritic arbor of identified entorhinal projection neurons was examined. The solitary apical dendrite of identified entorhinal projection neurons was prominently immunolabeled for GluR 5/6/7, as was the dendritic arbor of basilar dendrites of these neurons. The restriction of the large bulk of the dendritic arbor of identified entorhinal projection neurons to layer V implies that these neurons are likely to be heavily influenced by hippocampal output arriving in the deep layers of the entorhinal cortex. Immunoreactivity for GluR 5/6/7 throughout the dendritic arbor of such neurons indicates that this class of glutamate receptor is in a position to play a prominent role in mediating excitatory neurotransmission within hippocampal-entorhinal circuits.

TorsinA immunoreactivity in brains of patients with DYT1 and non-DYT1 dystonia.

A mutation of the DYT1 gene, which codes for torsinA, has been identified as the cause of one form of autosomal dominantly inherited dystonia. TorsinA immunohistochemistry was used to examine a case of DYT1, and several cases of non-DYT1, dystonia. No evidence was found for alterations of immunoreactivity at the light microscopic level, specifically neither cytoplasmic aggregations nor colocalization of torsinA immunoreactivity with a marker for endoplasmic reticulum. These findings contrast with results of recent cell culture studies of torsinA.

Manganese intoxication in the rhesus monkey: a clinical, imaging, pathologic, and biochemical study.

We gave three adult rhesus monkeys seven IV injections of manganese chloride at approximately 1-week intervals. We evaluated neurologic status by serial clinical examinations and performed a levodopa test if the animal developed features of basal ganglia dysfunction. After the animals were killed, we performed neuropathologic, neurochemical, and laser microprobe mass analysis (LAMMA) studies. Two of three animals developed a parkinsonian syndrome characterized by bradykinesia, rigidity, and facial grimacing suggestive of dystonia but not tremor. Neither animal responded to levodopa. Autopsy demonstrated gliosis primarily confined to the globus pallidus (GP) and the substantia nigra pars reticularis (SNr). We detected focal mineral deposits throughout the GP and SNr, particularly in a perivascular distribution. LAMMA studies noted that mineral deposits were primarily comprised of iron and aluminum. The severity of pathologic change correlated with the degree of clinical dysfunction. These studies demonstrate that, in contrast to Parkinson's disease (PD) and MPTP-induced parkinsonism, manganese primarily damages the GP and SNr and relatively spares the nigrostriatal dopaminergic system. Further, the results suggest that Mn-induced parkinsonism can be differentiated from PD and MPTP-induced parkinsonism by the clinical syndrome and response to levodopa. The accumulation of iron and aluminum suggests that iron/aluminum-induced oxidant stress may contribute to the damage associated with Mn toxicity.

Practical approaches to stereology in the setting of aging- and disease-related brain banks.

The unbiased sampling techniques of stereology have been developed to avoid the inaccuracies of using 'representative' sections for morphometric studies. In order to carry out a stereologically valid study, the region of interest must be fully available for sampling and its boundaries or its constituents must also be distinctly identifiable. However, in the setting of a brain bank in which only one half of the brain specimen is fixed for morphology, a logistic problem arises in satisfying the needs of the diagnostic neuropathologist with that of the stereologically oriented morphologist. We present a dissection approach in which the region for analysis must be used for unbiased sampling and also be available for paraffin-embedded neuropathologic work-up. Following fixation, a block consisting of the entire region of interest is removed intact and using a multibladed knife the block is subsectioned in the coronal plane at regular intervals. Alternate blocks are chosen for either paraffin embedding and destined to neuropathologic evaluation or are processed for stereology. The stereology blocks can be either cryoprotected or placed in phosphate buffer and are serially sectioned on a cryostat or a Vibratome. Preliminary analyses using this approach have provided reliable estimates of the total number of different neuronal populations and disease-related lesions in a variety of human and non-human banked specimens. In addition, this approach has definite advantages in that it provides rigorous quantitative estimates of neuropathologic changes that can be correlated to clinical data and does not compromise the routine neuropathological diagnostic procedure of the materials.

Aluminum, Alzheimer's disease, and the olfactory system.

In Alzheimer's disease, it has been recognized that there is a dramatic tendency for the development of neurofibrillary tangles among neurons of cortical regions associated with the olfactory system. We have demonstrated that neurofibrillary tangle-bearing neurons contain dramatically elevated levels of aluminum. The olfactory system, the only portion of the central nervous system with exposure to the external environment, is uniquely capable of uptake and transneuronal spread of exogenous substances. We argue that inasmuch as aluminum is not employed in any physiologic process, these deposits must arise from exogenous sources. Using parkinsonism-dementia complex of Guam as a model, we present data which suggest that the olfactory system is particularly vulnerable to damage and is affected very early in the disease. This supports the concept that etiologic agents of importance to this epidemic may be airborne in nature and may enter the central nervous system via the olfactory pathways.

Cholinergic deficits in the brains of patients with parkinsonism-dementia complex of Guam.

Patients with parkinsonism-dementia complex (PDC) of Guam showed moderate loss of choline acetyl transferase activity in the midfrontal and inferior parietal cortex, and severe loss in the superior temporal cortex. This deficit was similar to that seen in Alzheimer's disease and less severe than Lewy body disease. Thus, cholinergic deficits in the neocortex might contribute to some of the cognitive alterations in PDC of Guam.

Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminum in Parkinson's disease: a LAMMA study.

The Laser Microprobe Mass Analyzer (LAMMA) is a sensitive instrument for identifying and localizing trace elements in tissue samples. Using LAMMA, we have examined melanin-containing neurons of the substantia nigra in patients with Parkinson's disease (PD) and controls. We found that iron significantly accumulates within neuromelanin granules of patients with PD compared to controls. Increased aluminum was found in the neuromelanin granules of 2 of 3 PD cases but in no controls. The accumulation of iron and aluminum, which are known to promote oxidant stress, may account for the selective degeneration of neuromelanin-containing neurons in PD.

TorsinA accumulation in Lewy bodies in sporadic Parkinson's disease.

Parkinson's disease (PD) is a neurodegnerative disorder that is pathologically characterized by the presence of Lewy bodies in the brain. We show that Lewy bodies in PD are strongly immunoreactive for torsinA, the protein product of the DYT1 gene, which is associated with primary generalized dystonia. In the substantia nigra, torsinA immunoreactivity is localized to the periphery of Lewy bodies, whereas, in cortical Lewy bodies it is uniformly distributed. The significance of this finding is unknown, but may implicate torsinA in neuronal dysfunction that occurs in PD as well as in primary dystonia.

Organization and quantitative analysis of kainate receptor subunit GluR5-7 immunoreactivity in monkey hippocampus.

A monoclonal antibody specific for GluR5-7 (mAb-4F5) has been used to characterize the distribution of kainate class glutamate receptor subunits in monkey hippocampus. Immunolabeled neurons were present in all subfields of the hippocampus as well as the dentate gyrus and subiculum. Quantitative immunofluorescence analysis by confocal microscopy demonstrated differential levels of immunoreactivity such that the highest intensities were in neurons within CA1 and subiculum as compared with those within CA3 or dentate gyrus. The regional differences in levels of subunit immunoreactivity correlate with the relative vulnerability of hippocampal neurons in several neurodegenerative disorders.

A therapeutic trial of gangliosides and thymosin in the Wobbler mouse model of motor neuron disease.

Mixed bovine gangliosides have been reported to enhance neuronal regeneration and sprouting. The Wobbler mouse model of motor neuron disease was used to test the clinical effects of long-term ganglioside administration on the course of the disease. Mixed gangliosides were injected subcutaneously into a group of 5 Wobbler mice and compared to a control group of 5 Wobbler mice which received saline. Because of several reports implicating involvement of the immune system in ALS, a 3rd group of 5 Wobbler mice received thymosin. All mice were 4 weeks old at commencement of injections. The 3 groups were examined weekly and graded with respect to front leg power, ability to climb a vertical grating, and walking posture. After 4 months of treatment, no significant difference between either experimental group and the controls was found.

The association of aluminum Alzheimer's disease, and neurofibrillary tangles.

Alzheimer's disease is a progressive neurodegenerative disease characterized by the development of large numbers of neurofibrillary tangles in certain neuronal populations. Aluminum salts inoculated into experimental animals produce neurofilamentous lesions which are similar, but not identical, to the neurofibrillary tangle of Alzheimer's disease. Although a few reports suggest evidence of increased amounts of aluminum in the brains of Alzheimer's disease victims, such bulk analysis studies have been difficult to replicate. Using scanning electron microscopy with x-ray spectrometry, we have identified accumulations of aluminum in neurofibrillary tangle-bearing neurons of Alzheimer's disease. Similar accumulations have been identified in the neurofibrillary tangle-bearing neurons found in the brains of indigenous natives of Guam who suffer from parkinsonism with dementia and from amyotrophic lateral sclerosis. This ongoing research still cannot ascribe a causal role of aluminum in the pathogenesis of the neurofibrillary tangle; however, it does suggest that environmental factors may play an important part in the formation of this abnormality.

Aluminium and the neurofibrillary tangle: results of tissue microprobe studies.

Despite the contradictory results of studies attempting to compare the bulk brain tissue aluminium content of specimens from Alzheimer's disease patients and controls, microprobe studies from our laboratory have consistently documented evidence of selective accumulation of the element within the neurofibrillary tangle-bearing cells associated with this condition. Laser microprobe mass analysis (a highly sensitive and precise technique for trace elemental microprobe analysis) has now demonstrated that the most prominent aluminium accumulations occur within the neurofibrillary tangle itself. Similar findings have been obtained from microprobe studies of the neurofibrillary tangles which are a characteristic feature of amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam. Although the intraneuronal localization of aluminium in the Guam-derived specimens is similar to that of Alzheimer's disease, the concentration of aluminium is considerably higher than is encountered in Alzheimer's disease specimens. We conclude that aluminium is an integral component of the neurofibrillary tangle and raise the possibility that the cross-linking properties of this highly reactive metal may stabilize the constituent cytoskeletal proteins which make up this pathological structure.

Comparative techniques for determining cellular iron distribution in brain tissues.

Iron is essential for a number of normal brain functions and accumulates in high concentrations in specific regions of the brain. In pathological states, it may further accumulate in these and other areas that are typically low in iron content. The contribution of excess iron to potential central nervous system damage through its ability to donate an electron and to promote oxygen free radical formation has made the nature, location, extent, and process of iron deposition in the brain important areas of investigation. Nevertheless, there is relatively little information currently available on the cellular and subcellular distribution of iron in the central nervous system in either normal or diseased states. We describe and compare a number of the currently available techniques by which iron can be detected within the cellular components of the brain. Histochemical approaches, primarily in the form of the Perls' stain, yields information only on iron in its ferric state and is a relatively insensitive technique. Electron microscopy with x-ray spectrometry can provide positive identification of iron but has limitations regarding morphological verification of the specific cells being probed and also has a relatively high lowest detection limit. Secondary ion mass spectrometry and proton-induced x-ray spectrometry are both expensive, highly complex techniques with greater detection sensitivity, but they have problems identifying the cellular components being analyzed. Finally, laser microprobe mass analysis combines histological localization and identification of probe sites in plastic-embedded histological sections with detection limits in the single part per million range.(ABSTRACT TRUNCATED AT 250 WORDS)

Microprobe studies of aluminum accumulation in association with human central nervous system disease.

The neurofibrillary tangle, first described by Alzheimer in 1907, along with the senile plaque, represent the two principle neuropathologic lesions identified in the brains of patients with Alzheimer's disease. Aluminum salts inoculated into the central nervous system of certain experimental animals induces neurofilamentous lesions which are similar, but not identical to the neurofibrillary tangles seen in Alzheimer's disease. Although some reports provide evidence of increased amounts of aluminum in the brains of Alzheimer's disease victims, such bulk analysis studies have been difficult to replicate. Over the past several years we have approached this problem using a variety of microprobe techniques in order to determine the distribution of trace elements within individual nerve cells.Using scanning electron microscopy with X-ray spectrometry, we have identified accumulations of aluminum in the neurofibrillary tangle-bearing neurons of cases of Alzheimer's disease. Similar accumulations have been found in the brains of the indigenous natives of Guam who suffer from parkinsonism with dementia and from amyotrophic lateral sclerosis. Recent studies using laser microprobe mass analysis suggest that the predominance of aluminum identified within the parikaryon of neurofibrillary tangle-bearing neurons is located within the neurofibrillary tangle itself. Although this ongoing research still cannot ascribe a causal role for aluminum in the etiology and/or pathogenesis of the neurofibrillary tangle, the data suggest that certain environmental factors related to trace elemental constituents may play an important role in the formation of this type of cellular pathology.

Selective accumulation of aluminum and iron in the neurofibrillary tangles of Alzheimer's disease: a laser microprobe (LAMMA) study.

We report the results of an examination of the elemental content of neurofibrillary tangle-bearing and neurofibrillary tangle-free neurons identified within the hippocampus of 10 subjects with Alzheimer's disease and 4 neuropathologically intact age-matched control subjects. The study employed laser microprobe mass analysis (LAMMA), a technique that provides extremely sensitive multielement detection in plastic-embedded, semithin-sectioned tissues. Evidence for the selective accumulation of aluminum within the neurofibrillary tangle-bearing neurons was obtained in all 10 subjects with Alzheimer's disease. The site of aluminum deposition within these cells was the neurofibrillary tangle itself, and not the "nuclear region," as we previously reported. Iron accumulation was also detected within neurofibrillary tangles. Evaluation for the accumulation of other elements within the tangle-bearing neurons failed to reveal any other metallic element as being consistently present. In addition, probe sites directed to neurons identified in snap-frozen cryostat sections from 2 subjects with Alzheimer's disease revealed similar spectra with prominent aluminum-related peaks, confirming that our findings are not related to exogenous contamination through fixation, embedding, or other procedures prior to analysis. This study further confirms the association of aluminum and neurofibrillary tangle formation in Alzheimer's disease.

Evidence of neuronal oxidative damage in Alzheimer's disease.

Oxidative stress has been proposed as a pathogenetic mechanism in Alzheimer's disease. One mechanism of oxidative damage is the nitration of tyrosine residues in proteins, mediated by peroxynitrite breakdown. Peroxynitrite, a reaction product of nitric oxide and superoxide radicals, has been implicated in N-methyl-D-aspartate receptor-mediated excitotoxic damage. Reported evidence of oxidative stress in Alzheimer's disease includes increased iron, alterations in protective enzymes, and markers of oxidative damage to proteins and lipids. In this report, we demonstrate the presence of nitrotyrosine in neurofibrillary tangles of Alzheimer's disease. Nitrotyrosine was not detected in controls lacking neurofibrillary tangles. Immunolabeling was demonstrated to be specific nitrotyrosine in a series of control experiments. These observations link oxidative stress with a key pathological lesion of Alzheimer's disease, the neurofibrillary tangle, and demonstrate a pathogenetic mechanism in common with the other major neurodegenerative diseases of aging, Parkinson's disease and amyotrophic lateral sclerosis. These findings further implicate nitric oxide expression and excitotoxicity in the pathogenesis of cell death in Alzheimer's disease.

Investigation of a fatality from nonoccupational aluminum phosphide exposure: measurement of aluminum in tissue and body fluids as a marker of exposure.

This case study reports our clinical and laboratory investigation of the accidental death of a pregnant rural woman related to phosphine exposure from stored grain fumigated with aluminum phosphide (AIP3) pellets. Environmental data (housing proximity to fumigated grain and meteorologic conditions at the time) coupled with clinical data (tachycardia and the rapid development of pulmonary edema with no antecedent clinical abnormalities) suggested possible toxicant effects. Gross and microscopic autopsy findings demonstrated pulmonary edema. Because phosphine generated from the phosphide is highly reactive and unstable, our laboratory strategy for this investigation focused on the quantitative analysis of aluminum (AI) in blood, gastric contents, and in lung tissue to test the possibility of fumigant intoxication. We recovered 713 ng/ml Al from blood (normal laboratory range 2 to 42 ng/ml). Laser mass spectral analysis of lung tissue demonstrated high concentrations of Al in alveolar macrophages (> 200 ppm) with little or no Al in adjacent lung tissue. Control lung tissue from an urban autopsy case matched by age, sex, and smoking habits showed no demonstrable Al. We conclude that Al may be a useful marker for AIP3 exposure.

A laser microprobe mass analysis of trace elements in brain mineralizations and capillaries in Fahr's disease.

We report a detailed analysis of the content of aluminum, iron, zinc, copper, calcium, and magnesium in the non-vascular and pericapillary mineralizations and the normal capillaries of the globus pallidus and dentate nucleus of the cerebellum in two patients with clinically and neuropathologically confirmed Fahr's disease. The study employed laser microprobe mass analysis, a technique that enables highly sensitive detection of the levels of trace elements. In the globus pallidus, there was a significant increase in aluminum-, iron-, zinc-, and calcium-related peak intensity in the pericapillary and non-vascular mineralizations compared to the normal capillaries. The pericapillary and non-vascular mineralizations had comparable concentrations of these elements. No difference was found in copper levels between the different probe sites. Magnesium was almost absent in pericapillary mineralizations and normal capillaries, while it accumulated within non-vascular mineralizations. In the cerebellar dentate nucleus, non-vascular mineralizations displayed higher concentrations of all of these elements than normal capillaries, while pericapillary mineralizations had a higher aluminum and lower iron, copper, and calcium content than did non-vascular mineralizations. Zinc and magnesium were selectively deposited within the non-vascular mineralizations in this nucleus. Furthermore, the element composition of non-vascular mineralizations differed between the globus pallidus and dentate nucleus. These findings indicate that the formation of pericapillary and non-vascular mineralizations may be two independent phenomena which coexist in the course of Fahr's disease. The marked qualitative and quantitative differences in trace element content in non-vascular mineralizations between the globus pallidus and cerebellar dentate nucleus suggest that the involvement of trace elements in the pathogenesis of Fahr's disease is probably indirect.