Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2.

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.

Levels of soluble apolipoprotein E/amyloid-ß (Aß) complex are reduced and oligomeric Aß increased with APOE4 and Alzheimer disease in a transgenic mouse model and human samples.

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-ß (Aß) levels. Evidence suggests physical interactions between apoE and Aß are partially responsible for these functional effects. However, the apoE/Aß complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aß in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aß and an increase in soluble Aß, specifically oligomeric Aß (oAß), are associated with APOE4 and AD. Previously, soluble Aß42 and oAß levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aß levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aß levels isoform-specifically modulate soluble oAß clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aß levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aß levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aß42 levels decreased in AD patients compared with controls, oAß levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aß modulates oAß levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

APOE4-specific changes in Aß accumulation in a new transgenic mouse model of Alzheimer disease.

APOE4 is the greatest risk factor for Alzheimer disease (AD) and synergistic effects with amyloid-ß peptide (Aß) suggest interactions among apoE isoforms and different forms of Aß accumulation. However, it remains unclear how the APOE genotype affects plaque morphology, intraneuronal Aß, soluble Aß42, and oligomeric Aß (oAß), particularly in vivo. As the introduction of human APOE significantly delays amyloid deposition in transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), 5xFAD-Tg mice, which exhibit amyloid deposition by age 2 months, were crossed with apoE-targeted replacement mice to produce the new EFAD-Tg mice. Compared with 5xFAD mice, Aß deposition was delayed by ∼4 months in the EFAD mice, allowing detection of early changes in Aß accumulation from 2-6 months. Although plaque deposition is generally greater in E4FAD mice, E2/E3FAD mice have significantly more diffuse and E4FAD more compact plaques. As a first report in FAD-Tg mice, the APOE genotypes had no effect on intraneuronal Aß accumulation in EFAD mice. In E4FAD mice, total apoE levels were lower and total Aß levels higher than in E2FAD and E3FAD mice. Profiles from sequential three-step extractions (TBS, detergent, and formic acid) demonstrated that the lower level of total apoE4 is reflected only in the detergent-soluble fraction, indicating that less apoE4 is lipoprotein-associated, and perhaps less lipidated, compared with apoE2 and apoE3. Soluble Aß42 and oAß levels were highest in E4FAD mice, although soluble apoE2, apoE3, and apoE4 levels were comparable, suggesting that the differences in soluble Aß42 and oAß result from functional differences among the apoE isoforms. Thus, APOE differentially regulates multiple aspects of Aß accumulation.

Preferential interactions between ApoE-containing lipoproteins and Aß revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift.

The association between apolipoprotein E (apoE) and amyloid-ß peptide (Aß) may significantly impact the function of both proteins, thus affecting the etiology of Alzheimer's disease (AD). However, apoE/Aß interactions remain fundamentally defined by the stringency of the detection method. Here we use size exclusion chromatography (SEC) as a non-stringent approach to the detection of apoE/Aß interactions in solution, specifically apoE and both endogenous and exogenous Aß from plasma, CSF and astrocyte conditioned media. By SEC analysis, Aß association with plasma and CNS lipoproteins is apoE-dependent. While endogenous Aß elutes to specific human plasma lipoproteins distinct from those containing apoE, it is the apoE-containing lipoproteins that absorb excess amounts of exogenous Aß40. In human CSF, apoE, endogenous Aß and phospholipid elute in an almost identical profile, as do apoE, exogenous Aß and phospholipid from astrocyte conditioned media. Combining SEC fractionation with subsequent analysis for SDS-stable apoE/Aß complex reveals that apoE-containing astrocyte lipoproteins exhibit the most robust interactions with Aß. Thus, standardization of the methods for detecting apoE/Aß complex is necessary to determine its functional significance in the neuropathology characteristic of AD. Importantly, a systematic understanding of the role of apoE-containing plasma and CNS lipoproteins in Aß homeostasis could potentially contribute to identifying a plasma biomarker currently over-looked because it has multiple components.

Introducing Human APOE into Aß Transgenic Mouse Models.

Apolipoprotein E (apoE) and apoE/amyloid-ß (Aß) transgenic (Tg) mouse models are critical to understanding apoE-isoform effects on Alzheimer's disease risk. Compared to wild type, apoE(-/-) mice exhibit neuronal deficits, similar to apoE4-Tg compared to apoE3-Tg mice, providing a model for Aß-independent apoE effects on neurodegeneration. To determine the effects of apoE on Aß-induced neuropathology, apoE(-/-) mice were crossed with Aß-Tg mice, resulting in a significant delay in plaque deposition. Surprisingly, crossing human-apoE-Tg mice with apoE(-/-)/Aß-Tg mice further delayed plaque deposition, which eventually developed in apoE4/Aß-Tg mice prior to apoE3/Aß-Tg. One approach to address hAPOE-induced temporal delay in Aß pathology is an additional insult, like head injury. Another is crossing human-apoE-Tg mice with Aß-Tg mice that have rapid-onset Aß pathology. For example, because 5xFAD mice develop plaques by 2 months, the prediction is that human-apoE/5xFAD-Tg mice develop plaques around 6 months and 12 months before other human-apoE/Aß-Tg mice. Thus, tractable models for human-apoE/Aß-Tg mice continue to evolve.

Development and validation of a yeast high-throughput screen for inhibitors of Aß42 oligomerization.

Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid ß (Aß), as the primary toxic species in Alzheimer's disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aß(42) oligomer formation. Specifically, Aß(42) fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aß(42) oligomerization.

Preparing synthetic Aß in different aggregation states.

This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid-ß peptide (Aß). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer's disease (AD). Past decades of AD research highlight the dependence of Aß42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aß42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aß. To develop homogenous Aß42 assemblies, we initially monomerize the peptide, erasing any "structural history" that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aß42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aß42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aß42. We also present a protocol for fluorescently labeling oligomeric Aß42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections.

Cognitive effects of cell-derived and synthetically derived Aß oligomers.

Soluble forms of amyloid-ß peptide (Aß) are a molecular focus in Alzheimer's disease research. Soluble Aß dimers (≈8 kDa), trimers (≈12 kDa), tetramers (≈16 kDa) and Aß*56 (≈56 kDa) have shown biological activity. These Aß molecules have been derived from diverse sources, including chemical synthesis, transfected cells, and mouse and human brain, leading to uncertainty about toxicity and potency. Herein, synthetic Aß peptide-derived oligomers, cell- and brain-derived low-n oligomers, and Aß*56, were injected intracerebroventricularly (icv) into rats assayed under the Alternating Lever Cyclic Ratio (ALCR) cognitive assay. Cognitive deficits were detected at 1.3 µM of synthetic Aß oligomers and at low nanomolar concentrations of cell-secreted Aß oligomers. Trimers, from transgenic mouse brain (Tg2576), did not cause cognitive impairment at any dose tested, whereas Aß*56 induced concentration-dependent cognitive impairment at 0.9 and 1.3µM. Thus, while multiple forms of Aß have cognition impairing activity, there are significant differences in effective concentration and potency.

Overexpression of human apolipoprotein A-I preserves cognitive function and attenuates neuroinflammation and cerebral amyloid angiopathy in a mouse model of Alzheimer disease.

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-ß (Aß) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-ß precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aß in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aß-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.

Chain dynamics of nascent polypeptides emerging from the ribosome.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

Characterization of protein expression and folding in cell-free systems by maldi-tof mass spectrometry.

Recent advances in basic research, medicine, and biotechnology provide great motivation for the development of analytical tools to probe the behavior of target biomolecules in complex biological environments. Cell-free transcription-translation systems are an attractive medium for such studies, because they mimic several biochemical features of living cells, yet they are much more amenable to manipulation and spectroscopic analysis. However, few methods are currently available to characterize target proteins in cell-free systems. We have employed matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry for the detection and characterization of two cell-free expressed model proteins, cold shock protein A and apomyoglobin (apoMb) in cell-free systems. We exploited a combination of multiple selective isotope-labeling patterns for the identification of both full-length proteins and their in situ-generated proteolytic fragments. MALDI-TOF mass spectrometry-detected hydrogen/deuterium exchange, performed directly in the cell-free medium, allowed the assessment of apoMb's global degree of folding. The above methods are straightforward in that they do not require high levels of protein expression and allow the efficient characterization of both protein identity and global degree of folding.

Experimental and computational analysis of translation products in apomyoglobin expression.

This work focuses on the experimental analysis of the time-course of protein expression in a cell-free system, in conjunction with the development of a computational model, denoted as progressive chain buildup (PCB), able to simulate translation kinetics and product formation as a function of starting reactant concentrations. Translation of the gene encoding the apomyoglobin (apoMb) model protein was monitored in an Escherichia coli cell-free system under different experimental conditions. Experimentally observed protein expression yields, product accumulation time-course and expression completion times match with the predictions by the PCB model. This algorithm regards elementary single-residue elongations as apparent second-order events and it accounts for aminoacyl-tRNA regeneration during translation. We have used this computational approach to model full-length protein expression and to explore the kinetic behavior of incomplete chains generated during protein biosynthesis. Most of the observed incomplete chains are non-obligatory dead-end species, in that their formation is not mandatory for full-length protein expression, and that they are unable to convert to the expected final translation product. These truncated polypeptides do not arise from post-translational degradation of full-length protein, but from a distinct subpopulation of chains which expresses intrinsically more slowly than the population leading to full-length product. The PCB model is a valuable tool to predict full-length and incomplete chain populations and formulate experimentally testable hypotheses on their origin. PCB simulations are applicable to E.coli cell-free expression systems (both in batch and dialysis mode) under the control of T7 RNA polymerase and to other environments where transcription and translation can be regarded as kinetically decoupled.

In vitro expression and characterization of native apomyoglobin under low molecular crowding conditions.

The labile nature of membranes and organelles poses serious challenges to in situ biomolecule characterization in intact cells. Cell-free in vitro systems provide an alternative promising medium for the expression and characterization of protein conformation and function in a biochemical context that bears several similarities to the cellular environment. In addition, cell-free transcription-translation has recently emerged as a convenient method for protein selective isotope labeling, providing significant advantages for detailed NMR analysis. We report the cell-free expression of the model protein apomyoglobin (apoMb) in an Escherichia coli cell-free system and the effect of polyethylene glycol (PEG) on the expression yields. In contrast with in vivo protein production under control of the strong T7 promoter, apoMb is expressed in vitro in 100% soluble form. In-gel tryptic digestion followed by mass spectrometry were performed to confirm the protein identity. In order to probe the conformation of the newly expressed protein and investigate the feasibility of in situ structural analysis, high resolution protein characterization was carried out by 2D NMR spectroscopy. In vitro apoMb expression in a PEG-free environment is a convenient method for the production of soluble native-like protein under conditions amenable to selective isotopic labeling. Yields can be easily scaled-up by dialysis-assisted cell-free expression.

Painting protein misfolding in the cell in real time with an atomic-scale brush.

The direct observation of specific biochemical events in living cells is now possible as a result of combined advances in molecular biology and fluorescence microscopy. By genetically encoding the source of a unique spectroscopic signal, target proteins can be selectively detected within the complex cellular environment, with limited interference from background signals. A recent study takes advantage of arsenical reagent-based methodologies to monitor in vivo protein misfolding and inclusion body formation in real time. This approach promises to yield important information on the kinetics of aggregate formation in living cells and its relation to the time-course of protein expression and post-translational processing. The ability to follow protein self-association in real time accurately from its early stages is unique to this method, and has far-reaching implications for both biotechnology and misfolding-based disease.