False-Positive Error Rates for Reliable Digit Span and Auditory Verbal Learning Test Performance Validity Measures in Amnestic Mild Cognitive Impairment and Early Alzheimer Disease.

OBJECTIVE: The objective is to examine failure on three embedded performance validity tests [Reliable Digit Span (RDS), Auditory Verbal Learning Test (AVLT) logistic regression, and AVLT recognition memory] in early Alzheimer disease (AD; n = 178), amnestic mild cognitive impairment (MCI; n = 365), and cognitively intact age-matched controls (n = 206). METHOD: Neuropsychological tests scores were obtained from subjects participating in the Alzheimer's Disease Neuroimaging Initiative (ADNI). RESULTS: RDS failure using a ≤7 RDS threshold was 60/178 (34%) for early AD, 52/365 (14%) for MCI, and 17/206 (8%) for controls. A ≤6 RDS criterion reduced this rate to 24/178 (13%) for early AD, 15/365 (4%) for MCI, and 7/206 (3%) for controls. AVLT logistic regression probability of ≥.76 yielded unacceptably high false-positive rates in both clinical groups [early AD = 149/178 (79%); MCI = 159/365 (44%)] but not cognitively intact controls (13/206, 6%). AVLT recognition criterion of ≤9/15 classified 125/178 (70%) of early AD, 155/365 (42%) of MCI, and 18/206 (9%) of control scores as invalid, which decreased to 66/178 (37%) for early AD, 46/365 (13%) for MCI, and 10/206 (5%) for controls when applying a ≤5/15 criterion. Despite high false-positive rates across individual measures and thresholds, combining RDS ≤ 6 and AVLT recognition ≤9/15 classified only 9/178 (5%) of early AD and 4/365 (1%) of MCI patients as invalid performers. CONCLUSIONS: Embedded validity cutoffs derived from mixed clinical groups produce unacceptably high false-positive rates in MCI and early AD. Combining embedded PVT indicators lowers the false-positive rate.

AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.

The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 µg DNA per ml of media (21 µg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.

Fas-associated factor 1 and Parkinson's disease.

Fas-associated factor 1 or FAF1 is a Fas-binding protein implicated in apoptosis. FAF1 is the product of a gene at PARK 10 locus on chromosome 1p32, a locus associated with late-onset PD [Hicks, A.A., Petursson, H., Jonsson, T., Stefansson, H., Johannsdottir, H.S., Sainz, J., Frigge, M.L.et al., 2002. A susceptibility gene for late-onset idiopathic Parkinson's disease. Ann Neurol. 52, 549-555.]. In the present study we investigated the role of FAF1 in cell death and in Parkinson's disease (PD) pathogenesis. FAF1 levels were significantly increased in frontal cortex of PD as well as in PD cases with Alzheimer's disease (AD) pathology compared to control cases. Changes in FAF1 expression were specific to PD-related alpha-synuclein pathology and nigral cell loss. In addition, PD-related insults including, mitochondrial complex I inhibition, oxidative stress, and increased alpha-synuclein expression specifically increased endogenous FAF1 expression in vitro. Increased FAF1 levels induced cell death and significantly potentiated toxic effects of PD-related stressors including, oxidative stress, mitochondrial complex I inhibition and proteasomal inhibition. These studies, together with previous genetic linkage studies, highlight the potential significance of FAF1 in pathogenesis of idiopathic PD.