High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson's disease.

Alpha-synuclein seed amplification assays (αSyn-SAAs) are promising diagnostic tools for Parkinson's disease (PD) and related synucleinopathies. They enable detection of seeding-competent alpha-synuclein aggregates in living patients and have shown high diagnostic accuracy in several PD and other synucleinopathy patient cohorts. However, there has been confusion about αSyn-SAAs for their methodology, nomenclature, and relative accuracies when performed by various laboratories. We compared αSyn-SAA results obtained from three independent laboratories to evaluate reproducibility across methodological variations. We utilized the Parkinson's Progression Markers Initiative (PPMI) cohort, with DATSCAN data available for comparison, since clinical diagnosis of early de novo PD is critical for neuroprotective trials, which often use dopamine transporter imaging to enrich their cohorts. Blinded cerebrospinal fluid (CSF) samples for a randomly selected subset of PPMI subjects (30 PD, 30 HC, and 20 SWEDD), from both baseline and year 3 collections for the PD and HC groups (140 total CSF samples) were analyzed in parallel by each lab according to their own established and optimized αSyn-SAA protocols. The αSyn-SAA results were remarkably similar across laboratories, displaying high diagnostic performance (sensitivity ranging from 86 to 96% and specificity from 93 to 100%). The assays were also concordant for samples with results that differed from clinical diagnosis, including 2 PD patients determined to be clinically inconsistent with PD at later time points. All three assays also detected 2 SWEDD subjects as αSyn-SAA positive who later developed PD with abnormal DAT-SPECT. These multi-laboratory results confirm the reproducibility and value of αSyn-SAA as diagnostic tools, illustrate reproducibility of the assay in expert hands, and suggest that αSyn-SAA has potential to provide earlier diagnosis with comparable or superior accuracy to existing methods.

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

SOD1: a candidate gene for keratoconus.

PURPOSE: To screen superoxide dismutase 1 (SOD1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC). METHODS: Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the SOD1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced. RESULTS: A heterozygous genomic 7-base deletion in intron 2 of the SOD1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in 312 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn SOD (SOD1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon 3 (LE2E3). CONCLUSIONS: A unique genomic deletion within intron 2 close to the 5' splice junction of the SOD1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E3) that others have shown to code for proteins lacking the active site of the SOD1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.

Altered expression of aquaporins in bullous keratopathy and Fuchs' dystrophy corneas.

Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and beta2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease.

Expression of neuregulin 1, a member of the epidermal growth factor family, is expressed as multiple splice variants in the adult human cornea.

PURPOSE: To determine whether neuregulin 1 (Nrg-1) is expressed in the normal adult human cornea. METHODS: cDNA for Nrg-1 was obtained by direct amplification of RNA isolated from human corneal cell cultures. After sequencing, the likely exon/intron structure was determined by comparison to genomic sequence. RNA was purified from isolated corneal epithelium, corneal stroma, and primary cultures of both epithelial cells and stromal fibroblasts. Quantitative real-time polymerase chain reactions (qPCR) were performed to determine the overall levels of Nrg-1. A combination of fluorescent primers, restriction endonucleases, and image analysis was used to determine the proportion of each splice variant. Finally, the receptor family known to interact with Nrg-1 was examined to confirm its expression in corneal tissue. RESULTS: RT-PCR and Western blot analyses demonstrated that Nrg-1 and its receptor are expressed in adult corneal tissue and cultured cells derived from this tissue. qPCR suggested that epithelial cells and stromal cells produce equivalent levels of Nrg-1, but distinct variants were present that differ in proportion with each source of RNA. CONCLUSIONS: Eight distinct forms of Nrg-1 were expressed in the adult human cornea that differ by the alternate use of four exons. This altered the predicted coding sequence in three domains of Nrg-1. These domains are known to direct ligand/receptor interaction and the trafficking, processing, and release of Nrg-1 from the cell. Finally, there was a preference of exon usage that varied by location in the cornea and this pattern changed when cells were placed into culture.