α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies

Lewy bodies and Lewy neurites are the defining neuropathological characteristics of Parkinson’s disease and dementia with Lewy bodies. They are made of abnormal filamentous assemblies of unknown composition. We show here that Lewy bodies and Lewy neurites from Parkinson’s disease and dementia with Lewy bodies are stained strongly by antibodies directed against amino-terminal and carboxyl-terminal sequences of α-synuclein, showing the presence of full-length or close to full-length α-synuclein. The number of α-synuclein-stained structures exceeded that immunoreactive for ubiquitin, which is currently the most sensitive marker of Lewy bodies and Lewy neurites. Staining for α-synuclein thus will replace staining for ubiquitin as the preferred method for detecting Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method used for the extraction of paired helical filaments from Alzheimer’s disease brain. By immunoelectron microscopy, extracted filaments were labeled strongly by anti-α-synuclein antibodies. The morphologies of the 5- to 10-nm filaments and their staining characteristics suggest that extended α-synuclein molecules run parallel to the filament axis and that the filaments are polar structures. These findings indicate that α-synuclein forms the major filamentous component of Lewy bodies and Lewy neurites.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Parkinson disease: A new link between monoamine oxidase and mitochondrial electron flow

Two factors that contribute to the progression of Parkinson disease are a brain defect in mitochondrial respiration and the generation of hydrogen peroxide (H2O2) by monoamine oxidase (MAO). Here we show that the two are linked. Metabolism of the neurotransmitter dopamine, or other monoamines (benzylamine, tyramine), by intact rat brain mitochondria suppresses pyruvate- and succinate-dependent electron transport. MAO inhibitors prevent this action. Mitochondrial damage is also reversed during electron flow. A probable explanation is that MAO-generated H2O2 oxidizes glutathione to glutathione disulfide (GSSG), which undergoes thiol-disulfide interchange to form protein mixed disulfides, thereby interfering reversibly with thiol-dependent enzymatic function. In agreement with this premise, direct addition of GSSG to mitochondria resulted in similar reversible inhibition of electron transport. In addition, the monoamines induced an elevation in protein mixed disulfides within mitochondria. These observations imply that (i) heightened activity and metabolism of neurotransmitter by monoamine neurons may affect neuronal function, and (ii) apparent defects in mitochondrial respiration associated with Parkinson disease may reflect, in part, an established increase in dopamine turnover. The experimental results also target mitochondrial repair mechanisms for further investigation and may, in time, lead to newer forms of therapy.

Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains α-, β-, and γ-synuclein

Pathogenic α-synuclein (αS) gene mutations occur in rare familial Parkinson’s disease (PD) kindreds, and wild-type αS is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimer’s disease, but β-synuclein (βS) and γ-synuclein (γS) have not yet been implicated in neurological disorders. Here we show that in PD and DLB, but not normal brains, antibodies to αS and βS reveal novel presynaptic axon terminal pathology in the hippocampal dentate, hilar, and CA2/3 regions, whereas antibodies to γS detect previously unrecognized axonal spheroid-like lesions in the hippocampal dentate molecular layer. The aggregation of other synaptic proteins and synaptic vesicle-like structures in the αS- and βS-labeled hilar dystrophic neurites suggests that synaptic dysfunction may result from these lesions. Our findings broaden the concept of neurodegenerative “synucleinopathies” by implicating βS and γS, in addition to αS, in the onset/progression of PD and DLB.

Description of microcolumnar ensembles in association cortex and their disruption in Alzheimer and Lewy body dementias

The cortex of the brain is organized into clear horizontal layers, laminae, which subserve much of the connectional anatomy of the brain. We hypothesize that there is also a vertical anatomical organization that might subserve local interactions of neuronal functional units, in accord with longstanding electrophysiological observations. We develop and apply a general quantitative method, inspired by analogous methods in condensed matter physics, to examine the anatomical organization of the cortex in human brain. We find, in addition to obvious laminae, anatomical evidence for tightly packed microcolumnar ensembles containing approximately 11 neurons, with a periodicity of about 80 μm. We examine the structural integrity of this new architectural feature in two common dementing illnesses, Alzheimer disease and dementia with Lewy bodies. In Alzheimer disease, there is a dramatic, nearly complete loss of microcolumnar ensemble organization. The relative degree of loss of microcolumnar ensembles is directly proportional to the number of neurofibrillary tangles, but not related to the amount of amyloid-β deposition. In dementia with Lewy bodies, a similar disruption of microcolumnar ensemble architecture occurs despite minimal neuronal loss. These observations show that quantitative analysis of complex cortical architecture can be applied to analyze the anatomical basis of brain disorders.

A mitochondrial DNA clone is associated with increased risk for Alzheimer disease.

Severe mitochondrial genetic mutations lead to early degeneration of specific human tissues; milder mitochondrial mutations may cause degeneration at a later point in life. A mutation at position 4336 was reported to occur at increased frequency in individuals with Alzheimer disease (AD) and Parkinson disease [Shoffner, J. M., Brown, M. D., Torroni, A., Lott, M. T., Cabell, M. F., Mirra, S. S., Beal, M. F., Yang, C.-C., Gearing, M., Salvo, R., Watts, R. L., Juncos, J. L., Hansen, L. A., Crain, B. J., Fayad, M., Reckord, C. L. & Wallace, D. C. (1993) Genomics 17, 171-184]. We have investigated the notion that this mutation leads to excess risk of AD by using a case-control study design of 72 AD autopsies and 296 race- and age-matched controls. The 4336G mutation occurred at higher frequency in AD autopsies than age-matched controls, a statistically significant difference. Evolutionary analysis of mtDNAs bearing the 4336G mutation indicated they were more closely related to each other than to other mtDNAs, consistent with the model of a single origin for this mutation. The tight evolutionary relatedness and homoplasmy of mtDNAs that confer elevated risk for a late-onset disease contrast strikingly with the distant relatedness and heteroplasmy of mitochondrial genomes that cause early-onset disease. The dichotomy can be explained by a lack of selection against mutations that confer a phenotype at advanced age during most of the evolution of humans. We estimate that approximately 1.5 million Caucasians in the United States bear the 4336G mutation and are at significantly increased risk of developing mitochondrial AD in their lifetime. A mechanism for 4336G-mediated cell death is proposed.

Memory systems analyses of mnemonic disorders in aging and age-related diseases

The effects upon memory of normal aging and two age-related neurodegenerative diseases, Alzheimer disease (AD) and Parkinson disease, are analyzed in terms of memory systems, specific neural networks that mediate specific mnemonic processes. An occipital memory system mediating implicit visual-perceptual memory appears to be unaffected by aging or AD. A frontal system that may mediate implicit conceptual memory is affected by AD but not by normal aging. Another frontal system that mediates aspects of working and strategic memory is affected by Parkinson disease and, to a lesser extent, by aging. The aging effect appears to occur during all ages of the adult life-span. Finally, a medial-temporal system that mediates declarative memory is affected by the late onset of AD. Studies of intact and impaired memory in age-related diseases suggest that normal aging has markedly different effects upon different memory systems.

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.

d-β-Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

The heroin analogue 1-methyl-4-phenylpyridinium, MPP+, both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Aβ1–42, is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-β-hydroxybutyrate protected cultured mesencephalic neurons from MPP+ toxicity and hippocampal neurons from Aβ1–42 toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

An impairment in sniffing contributes to the olfactory impairment in Parkinson's disease

Although the presence of an olfactory impairment in Parkinson's disease (PD) has been recognized for 25 years, its cause remains unclear. Here we suggest a contributing factor to this impairment, namely, that PD impairs active sniffing of odorants. We tested 10 men and 10 women with clinically typical PD, and 20 age- and gender-matched healthy controls, in four olfactory tasks: (i) the University of Pennsylvania smell identification test; (ii and iii) detection threshold tests for the odorants vanillin and propionic acid; and (iv) a two-alternative forced-choice detection paradigm during which sniff parameters (airflow peak rate, mean rate, volume, and duration) were recorded with a pneomatotachograph-coupled spirometer. An additional experiment tested the effect of intentionally increasing sniff vigor on olfactory performance in 20 additional patients. PD patients were significantly impaired in olfactory identification (P < 0.0001) and detection (P < 0.007). As predicted, PD patients were also significantly impaired at sniffing, demonstrating significantly reduced sniff airflow rate (P < 0.01) and volume (P < 0.002). Furthermore, a patient's ability to sniff predicted his or her performance on olfactory tasks, i.e., the more poorly patients sniffed, the worse their performance on olfaction tests (P < 0.009). Finally, increasing sniff vigor improved olfactory performance in those patients whose baseline performance had been poorest (P < 0.05). These findings implicate a sniffing impairment as a component of the olfactory impairment in PD and further depict sniffing as an important component of human olfaction.