A genetic basis for the variable effect of smoking/nicotine on Parkinson's disease.

Prior studies have established an inverse association between cigarette smoking and the risk of developing Parkinson's disease (PD), and currently, the disease-modifying potential of the nicotine patch is being tested in clinical trials. To identify genes that interact with the effect of smoking/nicotine, we conducted genome-wide interaction studies in humans and in Drosophila. We identified SV2C, which encodes a synaptic-vesicle protein in PD-vulnerable substantia nigra (P=1 × 10(-7) for gene-smoking interaction on PD risk), and CG14691, which is predicted to encode a synaptic-vesicle protein in Drosophila (P=2 × 10(-11) for nicotine-paraquat interaction on gene expression). SV2C is biologically plausible because nicotine enhances the release of dopamine through synaptic vesicles, and PD is caused by the depletion of dopamine. Effect of smoking on PD varied by SV2C genotype from protective to neutral to harmful (P=5 × 10(-10)). Taken together, cross-validating evidence from humans and Drosophila suggests SV2C is involved in PD pathogenesis and it might be a useful marker for pharmacogenomics studies involving nicotine.

The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts.

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.

A neuropeptide gene defined by the Drosophila memory mutant amnesiac.

Mutations in genes required for associative learning and memory in Drosophila exist, but isolation of the genes has been difficult because most are defined by a single, chemically induced allele. Here, a simplified genetic screen was used to identify candidate genes involved in learning and memory. Second site suppressors of the dunce (dnc) female sterility phenotype were isolated with the use of transposon mutagenesis. One suppressor mutation that was recovered mapped in the amnesiac (amn) gene. Cloning of the locus revealed that amn encodes a previously uncharacterized neuropeptide gene. Thus, with the cloning of amn, specific neuropeptides are implicated in the memory process.

Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly Drosophila melanogaster. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.

Studying human neurodegenerative diseases in flies and worms.

Invertebrate models of several human neurodegenerative diseases have recently been described. These models faithfully replicate key neuropathological features of the human disorders. Because the basic cell biology of the nervous system is very similar in vertebrates and invertebrates, the sophisticated and rapid genetic analysis feasible in Drosophila and C. elegans promises significant insight into human neurodegenerative syndromes. In addition, the short lifespan, small size, and ease of culturing make worms and flies ideal for drug testing.

Neuropathologic overlap of progressive supranuclear palsy, Pick's disease and corticobasal degeneration.

Several neurodegenerative disorders contain tau-immunoreactive neuronal and glial inclusions throughout the cerebral cortex and brainstem. Although these diseases have been considered distinct clinicopathological entities, recent recognition of many neuropathological and clinical parallels has raised the question of overlap between the disorders. In addition, histopathological similarities sometimes complicate neuropathological diagnosis. To address these issues, we examined the morphology and differential distribution of pathologic lesions in three disorders: progressive supranuclear palsy, Pick's disease, and corticobasal degeneration. We found considerable similarity in the anatomical regions affected by the three entities; however, semiquantitative analysis revealed differential anatomical susceptibility. Similarly, although overlap existed in the morphology of tau-immunoreactive inclusions, characteristic differences remained and may be useful in differential diagnosis. In particular, glial inclusions varied dramatically between the disorders. Despite significant overlap among the three neurodegenerative diseases examined, the morphological and regional differences suggest that each is a distinct pathophysiological entity.

August 2000: Two cases with necrosis and hemorrhage in the putamen and white matter.

The August COM: Acute methanol poisoning is an uncommon, but well-recognized, cause of central nervous system injury. We present two autopsy cases showing the classic neuropathologic injuries in acute methanol poisoning: putamen and white matter necrosis and hemorrhage. In Case 1, putamen hemorrhages were striking; white matter pathology predominated in Case 2. The precise mechanism of methanol toxicity is unclear. Direct toxicity of metabolites, particularly formic acid, as well as ischemic injury and acidosis likely play a role. Methanol is readily available in many commercial products, and may be ingested accidentally or intentionally.

Neuropeptide modulation of learning and memory processes.

The ubiquitous nature of neuropeptides and their respective receptors in the central and peripheral nervous systems suggests that peptides play a key role in controlling physiological processes. Investigations on a cellular level have demonstrated that neuropeptides exert powerful modulatory effects on neurons and neuronal circuits; however, despite these compelling considerations, investigators have rarely been able to assign discrete functional roles to individual neuropeptides. Numerous studies have addressed the influence of neuropeptides on learning and memory processes. Workers have primarily utilized peripheral or central injection of neuropeptides to suggest a facilitatory, or less commonly inhibitory, role in acquisition, retention, or retrieval of memories. Although highly suggestive, critical concerns regarding the specificity of the observed effects have often remained. Recently, the neurogenetic approach has demonstrated the role of a novel neuropeptide in a specific memory phase, high affinity antagonists have confirmed the importance of some endogenous neuropeptides, and evidence of neuropeptide dysfunction in disease states, particularly Alzheimer's disease, has emerged. Continued refinement of traditional techniques, combined with information from alternative approaches, promises to consolidate the role of neuropeptides in learning and memory.

Cytoskeletal pathology in non-Alzheimer degenerative dementia: new lesions in diffuse Lewy body disease, Pick's disease, and corticobasal degeneration.

Increasing use of immunocytochemistry for evaluation of dementia disorders has revealed histopathological alterations that were previously unknown, even with sensitive silver techniques. Disorders [Pick's disease (PD), diffuse Lewy body disease (DLBD) and corticobasal degeneration (CBD)] in which immunocytochemistry has revealed occult pathology are discussed. All three disorders have neurofilament (NF) immunoreactive neuronal alterations in the neocortex. In DLBD round, eosinophilic cytoplasmic inclusions referred to as cortical Lewy bodies are neurofilament-positive, while in both PD and CBD neurofilament epitopes are expressed in irregularly swollen neurons and their proximal cell processes, which are referred to as ballooned neurons. Interestingly, the cortical neuronal population that is vulnerable to Lewy bodies is similar to that which is vulnerable to ballooned neurons. Furthermore, Lewy bodies can occasionally be detected within the cytoplasm of ballooned neurons. Besides neurofilament-immunoreactivity, Lewy bodies are immunoreactive for ubiquitin, while ballooned neurons are inconsistently stained with antibodies to ubiquitin. Both Lewy bodies and ballooned neurons can be appreciated with routine histology, but they are much easier to detect with immunocytochemistry. In contrast, a new type of neuritic alteration in the hippocampal CA2/3 region has been recognized in DLBD. These dystrophic neurites cannot be appreciated with routine histology and are only optimally seen with immunocytochemistry for ubiquitin. Their presence is a certain indication of the presence of cortical Lewy bodies. The microtuble associated protein tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD). Biochemical studies have shown that Pick bodies, argyrophilic neuronal inclusions that are highly characteristic of, if not pathognomonic for PD are also composed of abnormal tau protein. Along with Pick bodies, tau has recently been detected in glial cells in PD. Similar so-called "gliofibrillary tangles" are increasingly recognized in progressive supranuclear palsy. Previously, CBD was considered to be free of such lesions, but recent studies have revealed widespread tau-positive neuronal and glial cytoskeletal lesions in CBD. A distinctive type of tau-positive glial lesion in CBD is characterized by annular clusters of grain-like tau immunoreactivity reminiscent of a neuritic plaque in AD, except that the clusters are devoid of amyloid. The tau-positive profiles are consistently located around a central astrocyte cell body. Double labeling studies with glial fibrillary acidic protein, vimentin and CD44, which are markers for reactive astrocytes, demonstrates tau immunoreactivity within astrocytic processes; these "astrocytic plaques" appear to be specific for CBD. Although NF, ubiquitin and tau proteins are present in diverse neuronal and glial inclusions in these disorders, the morphology and distribution of these lesions differentiate non-AD dementias.

Rescue of the learning defect in dunce, a Drosophila learning mutant, by an allele of rutabaga, a second learning mutant.

rutabaga1 (rut1), a Drosophila learning mutant, has adenylate cyclase (EC 4.6.1.1) with reduced basal activity and the absence of calcium/calmodulin-stimulated activity. A second learning mutant, dunce, is defective in cyclic AMP degradation due to decreased or absent phosphodiesterase activity. These opposing biochemical defects allow rut1 to partially suppress the female sterility caused by elevated cyclic AMP levels in dunce flies. Selection of mutations that suppress dunce sterility has led to the isolation of two rutabaga alleles. The alleles (rut2 and rut3) decrease basal adenylate cyclase activity [Bellen, H. J., Gregory, B. K., Olsson, C. L. & Kiger, J. A. (1987) Dev. Biol. 121, 432-444] but, unlike the original rutabaga mutation, leave the calcium/calmodulin-stimulated activity intact. Behaviorally, the two alleles also differ from rut1. One of the mutations partially rescues the dunce learning defect, and flies bearing both alleles learn. Calcium responsiveness may thus be the crucial component of adenylate cyclase activity required for associative learning.

The synaptic vesicle protein synaptotagmin promotes formation of filopodia in fibroblasts.

Neuronal filopodia are actin-rich cytoplasmic extensions that are involved in motility and recognition in growth cones and maturing axonal endings. A detailed understanding of neuronal growth will depend on clarification of the membrane fusion events occurring during filopodial extension. The synaptic vesicle protein synaptotagmin seems to be intimately involved in exocytotic membrane fusion. Here we show that fibroblast cell lines transfected with synaptotagmin form long, highly branched, actin-rich filopodial processes, with the expressed synaptotagmin being incorporated into the plasma membrane. In contrast, cell lines expressing either of two other synaptic vesicle proteins, SV2 or synaptophysin, generate only rudimentary processes, and, like neurons, sort SV2 and synaptophysin to small intracellular vesicles. As presynaptic calcium entry regulates synaptic vesicle fusion, our results indicate that synaptotagmin might link neuronal activity with synaptic growth.

Neurodegenerative disorders with extensive tau pathology: a comparative study and review.

Many neurodegenerative disorders with onset in mid to late adult life present diagnostic challenges to clinicians and pathologists alike. A distinguishing neuropathological feature has traditionally been the presence or absence of neurofibrillary tangles. Recent biochemical and molecular biological studies have identified the microtubule-binding protein tau as the predominant component of these and related inclusions, and have provided powerful new reagents for the study of neurodegenerative diseases. Several diseases previously considered distinct pathophysiological entities contain similar tau-immunoreactive lesions, but qualitative and regional anatomical differences in vulnerability can differentiate the disorders. Comparison of tau-immunoreactive lesions in three relatively uncommon neurodegenerative diseases-progressive supranuclear palsy, Pick's disease, and corticobasal degeneration-illustrates the types of analyses that demonstrate unexpected pathological similarities, but also fundamental differences between these disorders. These results have important implications for the differential diagnosis of disorders containing tau-immunoreactive lesions, including Alzheimer's disease.

A Drosophila model of Parkinson's disease.

Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease and alpha-synuclein accumulates in Lewy bodies and Lewy neurites. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.

Widespread cytoskeletal pathology characterizes corticobasal degeneration.

Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial pathology. Using immunohistochemistry and laser confocal microscopy, we demonstrate that the nonamyloid cortical plaques of CBD are actually collections of abnormal tau in the distal processes of astrocytes. These glial cells express both vimentin and CD44, markers of astrocyte activation. Glial pathology also includes tau-positive cytoplasmic inclusions, here localized to Leu 7-expressing oligodendrocytes. In addition, a wide array of neuronal pathology is defined with tau-positive inclusions in multiple domains of a variety of cortical neurons. CBD thus exhibits widespread glial and neuronal cytoskeletal pathology, including a novel structure, the astrocytic plaque. CBD is a disease of generalized cytoskeletal disruption affecting several cell types and multiple domains of these cells. The further definition of CBD pathology refines the diagnosis and pathophysiological understanding of this unique disease and has important implications for other neurodegenerative diseases, like Alzheimer's disease, characterized by abnormal tau deposition.

Nerve sheath tumours with hybrid features of neurofibroma and schwannoma: a conceptual challenge.

AIMS: To characterize and delineate a subset of rare nerve sheath tumours showing hybrid features of neurofibroma and schwannoma. METHODS AND RESULTS: Nine lesions were identified in the authors' files showing predominant features of neurofibroma with distinct, often nodular regions of classical schwannomatous differentiation. Most patients were adults, eight out of nine were male. Of the nine lesions, two were dermal, two were subcutaneous and five were subfascial. Five lesions had a plexiform architecture and one patient had overt neurofibromatosis. One out of six patients with follow-up developed local recurrence. Schwannoma-like regions displayed strong S100 staining, in contrast to more varied and limited S100 reactivity in neurofibromatous areas. The Antoni A areas could be quite cellular with high MIB-1 proliferation indices. No lesion underwent malignant change. CONCLUSIONS: Our results demonstrate that some nerve sheath tumours may contain histologically clear components of both neurofibroma and schwannoma, suggesting that (despite evident and well-defined clinicopathological differences) these two lesions may be even more closely related than previously recognized. Whether this phenomenon results from a localized microenvironmental change or from a clonal genetic alteration remains unknown.

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.

Epitope expression and hyperphosphorylation of tau protein in corticobasal degeneration: differentiation from progressive supranuclear palsy.

Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial accumulation of abnormal tau protein. Using immunohistochemistry we analyzed tau epitope expression and phosphorylation state in CBD and compared them to cytoskeletal changes in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Epitopes spanning the entire length of the tau protein were present in CBD inclusions. An antibody against the alternatively spliced exon 3 did not recognize cytoskeletal lesions in CBD, but did in AD and PSP. Tau epitopes from each region of the molecule were present in cytoskeletal inclusions in CBD, including gray matter astrocytic plaques, gray and white matter threads, and oligodendroglial inclusions. As in AD, tau from CBD was highly phosphorylated. Antibodies that recognized phosphorylated tau epitopes reacted with material from CBD in a highly phosphatase-dependent manner. Again, all types of inclusions contained phosphorylated epitopes. We conclude that abnormal tau protein in CBD comprises the entire tau molecule and is highly phosphorylated, but is distinguished from AD and PSP by the paucity of epitopes contained in the alternatively spliced exon 3.