Janetta G. Culvenor

Dopamine promotes α-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway

Dopamine (DA) and α‐synuclein (α‐SN) are two key molecules associated with Parkinsons disease (PD). We have identified a novel action of DA in the initial phase of α‐SN aggregation and demonstrate that DA induces α‐SN to form soluble, SDS‐resistant oligomers. The DA:α‐SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with α‐SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron‐induced α‐SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of α‐SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.

The solubility of α-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing α‐synuclein (αSN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinsons disease (PD). In order to identify disease‐associated changes of αSN, this study compared the levels, solubility and molecular weight species of αSN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent‐soluble and detergent‐insoluble αSN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of αSN in the detergent‐soluble fraction of brain samples from pons and white matter but detergent‐insoluble αSN was not detected. There was an inverse correlation between buffered saline‐soluble and detergent‐soluble levels of αSN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of αSN between grey and white matter in disease may result from different processing of αSN in neurons compared with oligodendrocytes. Highly insoluble αSN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline‐soluble or detergent‐soluble forms of αSN are involved in the pathogenesis of other αSN‐related diseases.

Non-Aβ Component of Alzheimer's Disease Amyloid (NAC) Revisited: NAC and α-Synuclein Are Not Associated with Aβ Amyloid

α-Synuclein (αSN), also termed the precursor of the non-Aβ component of Alzheimers disease (AD) amyloid (NACP), is a major component of Lewy bodies and Lewy neurites pathognomonic of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). A fragment of αSN termed the non-Aβ component of AD amyloid (NAC) had previously been identified as a constituent of AD amyloid plaques. To clarify the relationship of NAC and αSN with Aβ plaques, antibodies were raised to three domains of αSN. All antibodies produced punctate labeling of human cortex and strong labeling of Lewy bodies. Using antibodies to αSN(75–91) to label cortical and hippocampal sections of pathologically proven AD cases, we found no evidence for NAC in Aβ amyloid plaques. Double labeling of tissue sections in mixed DLB/AD cases revealed αSN in dystrophic neuritic processes, some of which were in close association with Aβ plaques restricted to the CA1 hippocampal region. In brain homogenates αSN was predominantly recovered in the cytosolic fraction as a 16-kd protein on Western analysis; however, significant amounts of aggregated and αSN fragments were also found in urea extracts of SDS-insoluble material from DLB and PD cases. NAC antibodies identified an endogenous fragment of 6 kd in the cytosolic and urea-soluble brain fractions. This fragment may be produced as a consequence of αSN aggregation or alternatively may accelerate aggregation of the full-length αSN.

In Vitro Characterization of Pittsburgh Compound-B Binding to Lewy Bodies

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of α-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimers disease (AD), Aβ plaques are also present in most DLB cases. The contribution of Aβ to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Aβ burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to α-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human α-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to α-synuclein fibrils but with lower affinity than that demonstrated/reported for Aβ1–42 fibrils. Furthermore, [3H]-PIB was observed to bind to Aβ plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Aβ plaque-free (“pure DLB”). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Aβ plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Aβ plaques and not Lewy bodies.

Selective Inhibition of a Two-step Egress of Malaria Parasites from the Host Erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16–32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria‐infected red blood cells

Adhesion of parasite‐infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein‐3 (PfEMP3), is deposited under the membrane of the parasite‐infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.falciparum‐infected cell. Truncation of the C‐terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane‐associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite‐infected red blood cell.

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry.

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.

Biochemical aspects of the neuroprotective mechanism of PTEN-induced kinase-1 (PINK1).

Mutations in PTEN‐induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson’s disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial‐targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C‐terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor‐associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis.

α-Synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores

A growing body of evidence suggests that the non-Aβ component of Alzheimers disease amyloid precursor protein (NACP) or α-synuclein contributes to the neurodegenerative processes in Alzheimers disease (AD), Parkinsons disease (PD) and dementia with Lewy bodies (DLB). In the present study antisera to the N terminus and the NAC domain of the α-synuclein protein were employed to elucidate the expression pattern in brains of patients with AD, PD, DLB and control specimen. α-Synuclein exhibited an overall punctuate expression profile compatible with a synaptic function. Interestingly, while Lewy bodies were strongly immunoreactive, none of the α-synuclein antisera revealed staining in mature β-amyloid plaques in AD. These observations suggest that α-synuclein does not contribute to late neurodegenerative processes in AD brains.

Variable antigen associated with the surface of erythrocytes infected with mature stages of Plasmodium falciparum

Immune human sera were used to select a cDNA clone expressing an asexual blood-stage antigen of Plasmodium falciparum. Antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting and immunofluorescence. The antigen is present in mature-stage parasites as a high molecular weight protein of about 250 kDa and is apparently processed to smaller fragments in the merozoite. It varies in molecular weight and antibody reactivity in different isolates, and has been localized at the erythrocyte membrane by immunoelectronmicroscopy. Part of the protein is composed of exactly repeated hexapeptide units that constitute the strain-specific determinant. This molecule has similar characteristics to the strain-specific molecule believed to be responsible for cytoadherence.