Eric M. Norstrom

The Charge Structure of Helix 1 in the Prion Protein Regulates Conversion to Pathogenic PrPSc

ABSTRACT The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer (PrPSc) of the normal prion protein (PrPC). Accumulation of PrPSc occurs via a poorly defined process in which PrPSc complexes with and converts endogenous PrPC to nascent PrPSc. Recent experiments have focused on the highly charged first alpha helix (H1) of PrP. It has been proposed that two putative asparagine-to-arginine intrahelical salt bridges stabilize H1 in PrPC yet form intermolecular ionic bonds with adjacent PrP molecules during conversion of PrPC to PrPSc (M. P. Morrissey and E. I. Shakhnovich, Proc. Natl. Acad. Sci. USA 96:11293-11298, 1999). Subsequent work (J. O. Speare et al., J. Biol. Chem. 278:12522-12529, 2003 using a cell-free assay of PrPSc conversion suggested that rather than promoting conversion, the salt bridges stabilize PrPC against it. However, the role of individual H1 charges in PrPSc generation has not yet been investigated. To approach this question, we systematically reversed or neutralized each charged residue in H1 and tested the effect on conversion to PrPSc 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 PrPC. These data support the necessity of specific charge orientations in H1 for a productive PrPSc-PrPC complex.

Cytosolic Prion Protein Toxicity Is Independent of Cellular Prion Protein Expression and Prion Propagation

ABSTRACT Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrPSc), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrPC exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrPC is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp+/+) mice with PrP-ablated mice (TgPrnpo/o) to generate Tg1D4(Prnpo/o) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp+/+) and Tg1D4(Prnpo/o) mice, suggesting that cyPrP and PrPC function independently in the disease state. Additionally, Tg1D4(Prnpo/o) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrPSc. We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrPC and the generation of typical prion disease.

Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy

Prion diseases are linked to the accumulation of a misfolded isoform (PrPSc) of prion protein (PrP). Evidence suggests that lysosomes are degradation endpoints and sites of the accumulation of PrPSc. We questioned whether lysosomes participate in the early quality control of newly generated misfolded PrP. We found PrP carrying the disease-associated T182A mutation (Mut-PrP) was delivered to lysosomes in a Golgi-independent manner. Time-lapse live cell imaging revealed early formation and uptake of GFP-tagged Mut-PrP aggregates into LysoTracker labeled vesicles. Compared with Wt-PrP, Mut-PrP expression was associated with an elevation in several markers of the autophagy-lysosomal pathway, and it extensively colocalized with the autophagosome-specific marker, LC3B. In autophagy deficient (ATG5−/−) mouse embryonic fibroblasts, or in normal cells treated with the autophagy-inhibitor 3-MA, Mut-PrP colocalization with lysosomes was reduced to a similar extent. Additionally, 3-MA selectively impaired the degradation of insoluble Mut-PrP, resulting in an increase in protease-resistant PrP, whereas the induction of autophagy by rapamycin reduced it. These findings suggest that autophagy might function as a quality control mechanism to limit the accumulation of misfolded PrP that normally leads to the generation of PrPSc.

Live cell fluorescence resonance energy transfer predicts an altered molecular association of heterologous PrPSc with PrPC.

Prion diseases result from the accumulation of a misfolded isoform (PrPSc) of the normal host prion protein (PrPC). PrPSc propagates by templating its conformation onto resident PrPC to generate new PrPSc. Although the nature of the PrPSc-PrPC complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrPSc-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrPSc and recipient PrPC that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrPSc and PrPC mismatched at residue 129.

Identification and characterization of Aβ peptide interactors in Alzheimer’s disease by structural approaches

Currently, there are very limited pharmaceutical interventions for Alzheimer’s disease (AD) to alleviate the amyloid burden implicated in the pathophysiology of the disease. Alzheimer’s disease is characterized immunohistologically by the accumulation of senile plaques in the brain with afflicted patients progressively losing short-term memory and, ultimately, cognition. Although significant improvements in clinical diagnosis and care for AD patients have been made, effective treatments for this devastating disease remain elusive. A key component of the amyloid burden of AD comes from accumulation of the amyloid-beta (Aβ) peptide which comes from processing of the amyloid precursor protein (APP) by enzymes termed secretases, leading to production of these toxic Aβ peptides of 40–42 amino acids. New therapeutic approaches for reducing Aβ are warranted after the most logical avenues of inhibiting secretase activity appear less than optimal in ameliorating the progression of AD.Novel therapeutics may be gleaned from proteomics biomarker initiatives to yield detailed molecular interactions of enzymes and their potential substrates. Explicating the APPome by deciphering protein complexes forming in cells is a complementary approach to unveil novel molecular interactions with the amyloidogenic peptide precursor to both understand the biology and develop potential upstream drug targets. Utilizing these strategies we have identified EC 3.4.24.15 (EP24.15), a zinc metalloprotease related to neprilysin (NEP), with the ability to catabolize Aβ 1–42 by examining first potential in silico docking and then verification by mass spectrometry. In addition, a hormone carrier protein, transthyreitin (TTR), was identified and with its abundance in cerebrospinal fluid (CSF), found to clear Aβ by inhibiting formation of oligomeric forms of Aβ peptide. The confluence of complementary strategies may allow new therapeutic avenues as well as biomarkers for AD that will aid in diagnosis, prognosis and treatment.

Protein electrophoretic migration data from custom and commercial gradient gels.

This paper presents data related to the article “A method for easily customizable gradient gel electrophoresis” (A.J. Miller, B. Roman, E.M. Norstrom, 2016) [1]. Data is presented on the rate of electrophoretic migration of proteins in both hand-poured and commercially acquired acrylamide gradient gels. For each gel, migration of 9 polypeptides of various masses was measured upon completion of gel electrophoresis. Data are presented on the migration of proteins within separate lanes of the same gel as well as migration rates from multiple gels.

A method for easily customizable gradient gel electrophoresis.

Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE.

Impaired transmissibility of atypical prions from genetic CJDG114V

Objective To describe the clinicopathologic, molecular, and transmissible characteristics of genetic prion disease in a young man carrying the PRNP-G114V variant. Methods We performed genetic, histologic, and molecular studies, combined with in vivo transmission studies and in vitro replication studies, to characterize this genetic prion disease. Results A 24-year-old American man of Polish descent developed progressive dementia, aphasia, and ataxia, leading to his death 5 years later. Histologic features included widespread spongiform degeneration, gliosis, and infrequent PrP plaque-like deposits within the cerebellum and putamen, best classifying this as a Creutzfeldt-Jakob disease (CJD) subtype. Molecular typing of proteinase K-resistant PrP (resPrPSc) revealed a mixture of type 1 (∼21 kDa) and type 2 (∼19 kDa) conformations with only 2, rather than the usual 3, PrPSc glycoforms. Brain homogenates from the proband failed to transmit prion disease to transgenic Tg(HuPrP) mice that overexpress human PrP and are typically susceptible to sporadic and genetic forms of CJD. When subjected to protein misfolding cyclic amplification, the PrPSc type 2 (∼19 kDa) was selectively amplified. Conclusions The features of genetic CJDG114V suggest that residue 114 within the highly conserved palindromic region (113-AGAAAAGA-120) plays an important role in prion conformation and propagation.