The molecular hallmark of prion disease is the conversion of normal prion protein (PrP C ) to an insoluble, proteinase K-resistant, pathogenic isoform (PrP Sc ). Once generated, PrP Sc propagates by complexing with, and transferring its pathogenic conformation onto, PrP C . Defining the specific nature of this PrP Sc -PrP C interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrP Sc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (
Alzheimer’s disease (AD) is an age-related neurodegenerative disease and the most common form of dementia. AD is pathologically characterized by the deposition of pathogenic Aβ peptides that are derived from larger integral membrane proteins, termed β-amyloid precursor proteins (APPs). In an attempt to understand the function of APP, in vitro studies have focused on the identification of interacting proteins. To investigate the APP in vivo interactome in an unbiased manner, we generated mice that harbor a mouse prion protein promoter-driven cDNA encoding human APP-695 fused to a C-terminal affinity tag. Using this tag, we prepared mild detergent lysates from transgenic mouse brain cortical membrane preparations and isolated a number of previously identified APP-interacting proteins. In addition to these factors, mass spectrometric analysis revealed the presence of NEEP21 as a novel interacting protein. We now report that NEEP21 profoundly affects the processing of APP and Aβ production. Thus, this study demonstrates that using proteomic methods on our transgenic model can uncover important in vivo APP-interacting proteins that will provide insights into the biology of APP.
The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer ( Sc in scrapie-infected murine neuroblastoma (ScN2a) cells. We find that replacements of charged H1 residues with like charges permit conversion, while charge reversals hinder it. Neutralization of charges in the N-terminal (amino acids 143 to 146) but not the C-terminal (amino acids 147 to 151) half of H1 permits conversion, while complete reversal of charge orientation of the putative salt bridges produces a nonconvertible PrP. Circular dichroism spectroscopy studies and confocal microscopy immunofluorescence localization studies indicated that charge substitutions did not alter the secondary structure or cell surface expression of PrP C . These data support the necessity of specific charge orientations in H1 for a productive PrP Sc -PrP C complex.
Small with control: For miniaturization of protein aggregation experiments the interfacial chemistry must be controlled to avoid protein aggregation caused by interfacial adsorption. Plug-based microfluidics with defined surface chemistry (see schematic picture) can then be used to perform hundreds of aggregation experiments with volume-limited samples, such as cerebrospinal fluid from mice.
Alterations in amyloid beta precursor protein (APP) have been implicated in cognitive decline in Alzheimer's disease (AD), which is accelerated in Down syndrome/Trisomy 21 (DS/TS21), likely due to the extra copy of the APP gene, located on chromosome 21. Proteolytic cleavage of APP generates amyloid-β (Aβ) peptide, the primary component of senile plaques associated with AD. Reducing Aβ production is predicted to lower plaque burden and mitigate AD symptoms. Here, we designed a splice-switching antisense oligonucleotide (SSO) that causes skipping of the APP exon that encodes proteolytic cleavage sites required for Aβ peptide production. The SSO induced exon skipping in Down syndrome cell lines, resulting in a reduction of Aβ. Treatment of mice with the SSO resulted in widespread distribution in the brain accompanied by APP exon skipping and a reduction of Aβ. Overall, we show that an alternatively spliced isoform of APP encodes a cleavage-incompetent protein that does not produce Aβ peptide and that promoting the production of this isoform with an SSO can reduce Aβ in vivo. These findings demonstrate the utility of using SSOs to induce a spliced isoform of APP to reduce Aβ as a potential approach for treating AD.
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