Lysia S. Forno

Lewy bodies and parkinsonism in families with parkin mutations

Previous work has established that compound mutations and homozygous loss of function of the parkin gene cause early‐onset, autosomal recessive parkinsonism. Classically, this disease has been associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, without Lewy body pathology. We have sequenced the parkin gene of 38 patients with early‐onset Parkinsons disease (<41 years). Two probands with mutations were followed up. Clinical evaluation of their families was performed, blinded to both genetic and pathological findings. Chromosome 6q25.2‐27 haplotype analysis was carried out independently of the trait; parkin gene expression was examined at both the RNA and protein levels. Haplotype analysis of these families revealed a common chromosome 6, with a novel 40 bp exon 3 deletion that cosegregated with disease. In the proband of the smaller kindred, an exon 7 R275W substitution was identified in addition to the exon 3 deletion; RNA analysis demonstrated that the mutations were on alternate transcripts. However, Lewy body pathology typical of idiopathic Parkinsons disease was found at autopsy in the proband from the smaller kindred. These data suggest that compound heterozygous parkin mutations and loss of parkin protein may lead to early‐onset parkinsonism with Lewy body pathology, while a hemizygous mutation may confer increased susceptibility to typical Parkinsons disease.

Parkin Localizes to the Lewy Bodies of Parkinson Disease and Dementia with Lewy Bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkins E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.

Long-term effects of chronic methamphetamine administration in rhesus monkeys

Biochemical and neuropathological effects of exposure to a high dose regimen of methamphetamine were evaluated in rhesus monkeys approximately 4 years after the last drug injection. Concentrations of dopamine and serotonin in caudate were below control levels as were concentrations of serotonin in several other brain regions. These changes were more severe in a monkey that was exposed twice to the drug regimen. A decrease in caudate synaptosomal uptake of both neurotransmitters as well as neuropathological changes were evident in that monkey. Although it is possible that partial recovery occurred, these results strongly suggest that methamphetamine-induced neurotoxicity may be permanent.

Aging and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of dopaminergic neurons in the substantia nigra.

This study assessed the influence of aging on substantia nigra degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive neuronal degeneration was found in the substantia nigra of older (8-12 months of age) but not younger (6-8 weeks of age) mice given MPTP. Older mice did not have higher brain concentrations of either MPTP or 1-methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of MPTP, to account for the greater toxicity. In fact, older mice metabolized MPTP more rapidly than younger mice, probably because of the increase in monoamine oxidase activity that occurs with aging. Striatal synaptosomes from older mice did not accumulate more [3H]MPP+ than synaptosomes from younger mice. Thus, it is concluded that the greater neurodegenerative effect of MPTP in older animals is not due to greater levels or uptake of MPP+, but rather is related to a true increase in sensitivity of older dopaminergic cells to MPTP. For comparative purposes, the toxic effect of another dopaminergic neurotoxin, methamphetamine, was tested. Older animals were not more sensitive than young mature animals to the toxic effect of methamphetamine. This finding indicates that the increased sensitivity of older dopaminergic neurons to MPTP is selective. The link established here between aging and the neurodegenerative effect of MPTP, a toxin which produces parkinsonism in humans, provides a mechanism by which an age-related neurodegenerative disorder such as Parkinsons disease could be caused by an MPTP-like toxin in the environment.

Fate of nigrostriatal neurons in young mature mice given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: A neurochemical and morphological reassessment

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on nigrostriatal dopaminergic neurons in the mouse was re-examined in view of recent conflicting reports regarding the neurotoxic effect of MPTP in this experimental animal. It was found that while MPTP destroyed a substantial number of dopaminergic nerve terminals in the striatum of young mature (6-8 weeks old) mice, it left the majority of cells in the pars compacta of the substantia nigra (SNc) unaffected. It was also found that 5 months after MPTP treatment there was substantial, although incomplete, recovery of striatal DA nerve terminal markers (DA level, metabolites, uptake, [3H]mazindol binding). Given these observations, it is concluded that while the young mature MPTP mouse may not be a valid animal model of Parkinsons disease (since it does not develop severe SNc cell loss characteristic of this disorder), it will be valuable for the study of how MPTP destroys dopaminergic nerve terminals and may prove useful as an experimental system for studying recovery of dopaminergic fibers after injury and for exploring ways to accelerate this recovery.

Ultrastructure of lewy bodies in the stellate ganglion

SummaryThe Lewy body, a characteristic nerve cell inclusion in idiopathic parkinsonism, was examined by electron microscopy in the stellate ganglion, obtained from 9 patients at autopsy. Three main forms of Lewy bodies or Lewy body-related structures were demonstrated: A. Rare filamentous Lewy bodies, similar to Lewy bodies in the central nervous system. B. Granular Lewy bodies in nerve cell processes. C. Abnormal nerve cell processes, filled with heterogenous material. Large dense core vesicles were prominent in the last 2 forms. None of these abnormalities were found in 2 control groups consisting of 9 parkinsonism cases without central nervous system Lewy bodies, and 17 cases without parkinsonism.The filamentous Lewy body (type A) was found in the perikaryon and was surrounded by neuromelanin, whereas the other forms (type B and C) were seen in nerve cell processes.Mitochondrial inclusions, present mainly, but not exclusively, in neuromelanin-containing cells, were not related to Lewy body formation or to parkinsonism.

Reaction of the substantia nigra to massive basal ganglia infarction

SummaryThe human substantia nigra can react to destruction of the basal ganglia in several ways. In ten brains with massive unilateral infarction of the basal ganglia slight to moderate nerve cell loss was present in the ipsilateral substantia nigra. The severe nerve cell loss reported in mostly young experimental animals was not observed. One case also displayed a fine network of myelinated and unmyelinated fibers surrounding pigmented nerve cells and dendrites in the ispilateral substantia nigra. Electron microscopy of the substantia nigra from this case showed neurofilamentous hyperplasia, paired helical filaments and rare straight filaments, but only on the side ipsilateral to the striatal infarct.The nerve cell loss in the ten cases was interpreted as a mainly retrograde degeneration, the perineuronal sprouts in case 10 as a reaction to partial deafferentation, and the paired helical filaments as either a retrograde or a transsynaptic reaction in the substantia nigra ipsilateral to the basal ganglia destruction.

Immunohistochemical localization of choline acetyl-transferase in the human cerebellum

Guinea pig antiserum specific to purified bovine choline acetyltransferase was found to cross-react with human enzyme. The peroxidase-antiperoxidase immunohistochemical method was then used to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded human cerebellum from normal as well as from Huntingtons disease brains. Choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. These immunohistochemical findings reveal the distribution of cholinergic axons and their terminals. The results are not only similar to our previous studies using the same method on the localization of choline acetyltransferase in rabbit cerebellum, but also demonstrate that some mossy fibers are cholinergic as suggested by others.

Pick bodies in the locus ceruleus

SummaryIn classical Picks disease with typical Pick bodies, inclusions resembling those present in the cerebral cortex are frequently found in the locus ceruleus. In three such cases Pick bodies were studied by light and electron microscopy and compared with Lewy bodies, inclusions more commonly found in this location. In contrast to the situation in the cerebral cortex, nerve cells with multiple Pick bodies were often found in the locus ceruleus, but in other respects definite light and electron microscopic differences between Pick bodies and Lewy bodies were present. Pick bodies were slightly basophilic and never had a central core or a peripheral halo. They were intensely argyrophilic. Differences in immunocytochemical reactions were especially marked with antibodies to tau and to paired helical filaments. Pick bodies displayed an intense reaction with these two antibodies, contrasting with that of Lewy bodies, which either lacked reactivity or reacted in a peripheral band. By electron microscopy the Pick bodies were composed of random filaments with smooth contour, whereas typical Lewy bodies had fuzzy deposits on filaments that radiated from a central core. Pick bodies in the locus ceruleus therefore maintained their immunocytochemical and electron microscopic characteristics and did not take on the character of Lewy bodies. Such differences point to a different pathogenesis and perhaps etiology of these two types of inclusions and attest to the marked difference clinically and pathologically between Picks and Parkinsons diseases.

Synaptic morphology in the human locus ceruleus

SummaryWe have studied the synaptic ultrastructure in human autopsy material from the locus ceruleus, an important noradrenergic center. Ten cases of Alzheimers disease, ten cases of Parkinsons disease, and ten control brains were examined.Only a few differences in synaptic morphology between the three groups were found. Multiple symmetrical and asymmetrical contacts on medium-sized dendrites were characteristically present. Axosomatic synapses were also common. In Alzheimers disease axosomatic synapses on nerve cell perikarya containing neurofibrillary tangles, and in Parkinsons disease on perikarya containing Lewy bodies, could be demonstrated. Dendritic spines were rare, but the control group had structures interpreted as “minispines”. In agreement with studies in the rat, cat, and rabbit, no spine apparatus was present, but in contrast to those studies no somatic spines were identified. Rare axoaxonic synapses were found in the control group. Vesicle content was often pleomorphic with flattened vesicles.Large dense core vesicles were present in variable numbers in nerve cell processes, and large accumulations of such vesicles were seen in two thirds of our cases, most abundant in the Alzheimers and Parkinsons disease groups. Such terminals may be aminergic, perhaps serotonergic, and may be a normal component of the locus ceruleus. Their greater abundance in Alzheimers and Parkinsons disease may be due to accumulation of the amines in afferent terminals, which have been deprived of their postsynaptic connections due to the degenerative disease process.