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.

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.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Investigation by Parkinson’s Disease Research Group of United Kingdom into excess mortality seen with combined levodopa and selegiline treatment in patients with early, mild Parkinson’s disease: further results of randomised trial and confidential inquiry

Objective: To determine whether the excess mortality observed in patients who received both levodopa and selegiline in a randomised trial could be explained by revised diagnosis of Parkinson’s disease, autonomic or cardiovascular effects, more rapid disease progression, or drug interactions.

Neuroprotective strategy for Alzheimer disease: intranasal administration of a fatty neuropeptide.

Neurodegenerative diseases, in which neuronal cell disintegrate, bring about deteriorations in cognitive functions as is evidenced in millions of Alzheimer patients. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective and to play an important role in the acquisition of learning and memory. A potent lipophilic analogue to VIP now has been synthesized, [stearyl-norleucine17]VIP ([St-Nle17]VIP), that exhibited neuroprotection in model systems related to Alzheimer disease. The beta-amyloid peptide is a major component of the cerebral amyloid plaque in Alzheimer disease and has been shown to be neurotoxic. We have found a 70% loss in the number of neurons in rat cerebral cortical cultures treated with the beta-amyloid peptide (amino acids 25-35) in comparison to controls. This cell death was completely prevented by cotreatment with 0.1 pM [St-Nle17]VIP. Furthermore, characteristic deficiencies in Alzheimer disease result from death of cholinergic neurons. Rats treated with a cholinergic blocker (ethylcholine aziridium) have been used as a model for cholinergic deficits. St-Nle-VIP injected intracerebroventricularly or delivered intranasally prevented impairments in spatial learning and memory associated with cholinergic blockade. These studies suggest both an unusual therapeutic strategy for treatment of Alzheimer deficiencies and a means for noninvasive peptide administration to the brain.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson’s disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP+). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.

Mortality in people taking selegiline: observational study

Objective: To evaluate mortality among patients with Parkinson’s disease receiving different treatment.

Intrinsic responses to Borna disease virus infection of the central nervous system

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs for egr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor α, macrophage inflammatory protein 1β, interleukin 6, and mob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.

Density-dependent decline of host abundance resulting from a new infectious disease

Although many new diseases have emerged within the past 2 decades [Cohen, M. L. (1998) Brit. Med. Bull. 54, 523–532], attributing low numbers of animal hosts to the existence of even a new pathogen is problematic. This is because very rarely does one have data on host abundance before and after the epizootic as well as detailed descriptions of pathogen prevalence [Dobson, A. P. & Hudson, P. J. (1985) in Ecology of Infectious Diseases in Natural Populations, eds. Grenfell, B. T. & Dobson, A. P. (Cambridge Univ. Press, Cambridge, U.K.), pp. 52–89]. Month by month we tracked the spread of the epizootic of an apparently novel strain of a widespread poultry pathogen, Mycoplasma gallisepticum, through a previously unknown host, the house finch, whose abundance has been monitored over past decades. Here we are able to demonstrate a causal relationship between high disease prevalence and declining house finch abundance throughout the eastern half of North America because the epizootic reached different parts of the house finch range at different times. Three years after the epizootic arrived, house finch abundance stabilized at similar levels, although house finch abundance had been high and stable in some areas but low and rapidly increasing in others. This result, not previously documented in wild populations, is as expected from theory if transmission of the disease was density dependent.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. In Arabidopsis thaliana, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen Pseudomonas syringae pv. tomato (Pst). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against Pst. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated Npr1 transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against Pst. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.

Inhibition of the ubiquitin-proteasome system in Alzheimer's disease

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub+1, has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub+1 expression should be deleterious. Here we show that Ub+1 is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub+1 in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.

Abrogation of disease development in plants expressing animal antiapoptotic genes

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant–pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host–cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant–pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule.

Graves disease is an autoimmune thyroid disease characterized by the presence of antibodies against the thyrotropin receptor (TSHR), which stimulate the thyroid to cause hyperthyroidism and/or goiter. By immunizing mice with fibroblasts transfected with both the human TSHR and a major histocompatibility complex class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of Graves disease: stimulating TSHR antibodies, thyrotropin binding inhibiting immunoglobulins, which are different from the stimulating TSHR antibodies, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen. The results suggest that the aberrant expression of major histocompatibility complex class II molecules on cells that express a native form of the TSHR can result in the induction of functional anti-TSHR antibodies that stimulate the thyroid. They additionally suggest that the acquisition of antigen-presenting ability on a target cell containing the TSHR can activate T and B cells normally present in an animal and induce a disease with the major features of autoimmune Graves.