Amelia McGlade

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.

Acute exposure to prion infection induces transient oxidative stress progressing to be cumulatively deleterious with chronic propagation in vitro

Neuronal loss is a pathological feature of prion diseases for which increased reactive oxygen species (ROS) and consequent oxidative stress is one proposed mechanism. The processes underlying ROS production in prion disease and the precise relationship to misfolding of the prion protein remain obscure. Using cell culture models of prion infection we found that cells demonstrate a rapid, prion protein (PrP) dependent, increase in intracellular ROS following exposure to infectious inoculum. ROS production correlated with internalisation and increased intracellular protease resistant PrP (PrP(Res)). The ROS increase was predominantly lysosomal in origin but not sustained, with cells adapting within 48 hours. Overall ROS levels remained normal in the chronically prion infected cell population; however a subpopulation characterised by loss of membrane phosphatidylserine asymmetry exhibited highly peroxidised intracellular aggregates that localised with PrP and intense caspase activation. These apoptotic cells showed increased ROS closely correlating with increased PrP(Res). Our findings demonstrate that a PrP-dependent, transient, increase in intracellular ROS is characteristic of acute cellular prion infection, while chronic phases of prion infection in vitro are associated with a significant subpopulation manifesting apoptosis accompanying heightened oxidative stress and increased PrP(Res) burden. Such observations strengthen the direct links between heightened ROS and ongoing prion propagation with eventual cellular demise.

MEK1 transduces the prion protein N2 fragment antioxidant effects.

The prion protein (PrPC) when mis-folded is causally linked with a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies or prion diseases. PrPC normal function is still incompletely defined with such investigations complicated by PrPC post-translational modifications, such as internal cleavage, which feasibly could change, activate, or deactivate the function of this protein. Oxidative stress induces β-cleavage and the N-terminal product of this cleavage event, N2, demonstrates a cellular protective response against oxidative stress. The mechanisms by which N2 mediates cellular antioxidant protection were investigated within an in vitro cell model. N2 protection was regulated by copper binding to the octarepeat domain, directing the route of internalisation, which stimulated MEK1 signalling. Precise membrane interactions of N2, determined by copper saturation, and involving both the copper-co-ordinating octarepeat region and the structure conferred upon the N-terminal polybasic region by the proline motif, were essential for the correct engagement of this pathway. The phenomenon of PrPC post-translational modification, such as cleavage and copper co-ordination, as a molecular “switch” for activation or deactivation of certain functions provides new insight into the apparent multi-functionality of PrPC.

No evidence for prion protein gene locus multiplication in Creutzfeldt-Jakob disease

Precedent of causative multiplication of key gene loci exists in familial forms of both Alzheimers and Parkinsons diseases. Genetic Creutzfeldt-Jakob disease (CJD) is often clinically indistinguishable from sporadic disease and inexplicably, a negative family history of a similar disorder occurs in around 50-90% of patients harboring the most common, disease-associated, prion protein gene (PRNP) mutations. We undertook semi-quantitative analysis of the PRNP copy number in 112 CJD patients using quantitative polymerase chain reaction. All included cases satisfied classification criteria for probable or definite sporadic CJD, ascertained as part of longstanding, prospective, national surveillance activities. No examples of additional copies of the PRNP locus as an explanation for their disease was found in any of the 112 sporadic CJD patients. Hence, contrasting with more common, age-related neurodegenerative diseases, the genetic aetiology in human prion disease continues to appear entirely restricted to small scale mutations within a single gene, with no evidence of multiplication of this validated candidate gene locus as a cause.

14-3-3 protein detection and sporadic CJD: the status quo serves well while awaiting progress

Of the neurodegenerative disorders, prion diseases pose some unique challenges. The phenotypic spectrum is sufficiently diverse that this diagnosis will not infrequently intrude as a consideration, with the attendant risk of inadvertent disease transmission during healthcare provision adding concerns not extant in relation to most neurological diseases, especially disorders such as Alzheimers disease. Prompt, accurate diagnosis can assist in pre-empting and thereby minimising transmission risk, while serving to avoid unhelpful investigations and treatments, and facilitate more informed prognostication. Notwithstanding the acknowledged need to continue searching for improvements in our diagnostic capacity, there has fortunately been major progress over the last 10–15 years in relation to the premortem diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD), the most common form of human prion disease. In addition to the more recent contribution of brain MRI,1 the recognition that 14-3-3 protein detection in …

Is a microRNA-328 binding site in PAX6 associated with Rolandic epilepsy?

Is a microRNA-328 binding site in PAX6 associated with Rolandic epilepsy? Amelia McGlade, Kenneth A. Myers, Samuel F. Berkovic, Ingrid E. Scheffer, Slav e Petrovski & Michael S. Hildebrand* Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia Florey Institute and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

Genetic prion disease-associated myelodysplasia and SIADH in siblings.

F. C. F. Chang, Y. Berman, M. E. Buckland, N. MacKinlay, A. McGlade, S. Collins and K. Ng Department of Neurology, Department of Clinical Genetics, Royal North Shore Hospital and the University of Sydney, Sydney, NSW; Department of Neuropathology, Royal Prince Alfred Hospital and the University of Sydney, Sydney, NSW; Department of Haematology, Royal North Shore Hospital and the University of Sydney, Sydney, NSW; and Australian National CJD Registry, Department of Pathology, The University of Melbourne, Parkville, VIC, Australia

KANSL1 variation is not a major contributing factor in self-limited focal epilepsy syndromes of childhood

Background KANSL1 haploinsufficiency causes Koolen-de Vries syndrome (KdVS), characterized by dysmorphic features and intellectual disability; amiable personality, congenital malformations and seizures also commonly occur. The epilepsy phenotypic spectrum in KdVS is broad, but most individuals have focal seizures with some having a phenotype resembling the self-limited focal epilepsies of childhood (SFEC). We hypothesized that variants in KANSL1 contribute to pathogenesis of SFEC. Materials and methods We screened KANSL1 for single nucleotide variants in 90 patients with SFEC. We then screened a cohort of 208 patients with two specific SFEC syndromes, childhood epilepsy with centrotemporal spikes (CECTS) and atypical childhood epilepsy with centrotemporal spikes (ACECTS) for KANSL1 variants. The second cohort was also used to evaluate minor allelic variants that appeared overrepresented in the initial cohort. Results One variant, p.Lys104Thr, was predicted damaging and appeared overrepresented in our 90-patient cohort compared to Genome Aggregation Database (gnomAD) allele frequency (0.217 to 0.116, with no homozygotes in gnomAD). However, there was no difference in p.Lys104Thr allele frequency in the follow-up CECTS/ACECTS cohort and controls. Four rare KANSL1 variants of uncertain significance were identified in the CECTS/ACECTS cohort. Discussion Our data do not support a major role for KANSL1 variants in pathogenesis of SFEC.