PET Radiopharmaceuticals for Alzheimer's Disease and Parkinson's Disease Diagnosis, the Current and Future Landscape.

Ironically, population aging which is considered a public health success has been accompanied by a myriad of new health challenges, which include neurodegenerative disorders (NDDs), the incidence of which increases proportionally to age. Among them, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common, with the misfolding and the aggregation of proteins being common and causal in the pathogenesis of both diseases. AD is characterized by the presence of hyperphosphorylated τ protein (tau), which is the main component of neurofibrillary tangles (NFTs), and senile plaques the main component of which is ß-amyloid peptide aggregates (Aß). The neuropathological hallmark of PD is α-synuclein aggregates (α-syn), which are present as insoluble fibrils, the primary structural component of Lewy body (LB) and neurites (LN). An increasing number of non-invasive PET examinations have been used for AD, to monitor the pathological progress (hallmarks) of disease. Notwithstanding, still the need for the development of novel detection tools for other proteinopathies still remains. This review, although not exhaustively, looks at the timeline of the development of existing tracers used in the imaging of Aß and important moments that led to the development of these tracers.

A Novel Dual Fluorochrome Near-Infrared Imaging Probe for Potential Alzheimer's Enzyme Biomarkers-BACE1 and Cathepsin D.

A molecular imaging probe to fluorescently image the ß-site of the amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes associated with Alzheimer's disease (AD) was designed and synthesized. This imaging probe was built upon iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, or CLIO). Peptide substrates containing a terminal near-infrared fluorochrome (fluorophore emitting at 775 nm for CatD or fluorophore emitting at 669 nm for BACE1) were conjugated to the CLIO nanoparticles. The CatD substrate contained a phenylalanine-phenylalanine cleavage site more specific to CatD than BACE1. The BACE1 substrate contained the sequence surrounding the leucine-asparagine cleavage site of the BACE1 found in the Swedish mutation of APP, which is more specific to BACE1 than CatD. These fluorescently-labeled peptide substrates were then conjugated to the nanoparticle. The nanoparticle probes were purified by gel filtration, and their fluorescence intensities were determined using a fluorescence plate reader. The CatD peptide substrate demonstrated a 15.5-fold increase in fluorescence when incubated with purified CatD enzyme, and the BACE1 substrate exhibited a 31.5-fold increase in fluorescence when incubated with purified BACE1 enzyme. Probe specificity was also demonstrated in the human H4 neuroglioma cells and the H4 cells stably transfected with BACE1 in which the probe monitored enzymatic cleavage. In the H4 and H4-BACE1 cells, BACE1 and active CatD activity increased, an occurrence that was reflected in enzyme expression levels as determined by immunoblotting. These results demonstrate the applicability of this probe for detecting potential Alzheimer's enzyme biomarkers.

New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies.

The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the ß-amyloid protein (Aß) in Alzheimer's disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies.

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

High expression and purification of amino-terminal fragment of human amyloid precursor protein in Pichia pastoris and partial analysis of its properties.

The cleaved amino-terminal fragment of human amyloid precursor protein (N-APP) binds death receptor 6 (DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer's disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, obtaining abundant and purified N-APP is fundamental and critical. The recombinant N-APP has been produced in mammalian expression system. However, the cost and yield disadvantages of mammalian expression system make it less ideal for protein mass production. Here, we successfully expressed and purified recombinant N-terminal 18-285 amino acid residues of human amyloid precursor protein from the methylotrophic yeast Pichia pastoris with a high yield of 50 mg/L. Flow cytometry indicated the purified N-APP-induced obvious apoptosis of human neuroblastoma SHEP cells.

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein.

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization.

Contribution of CE to the analysis of protein or peptide biomarkers.

Biomarker analysis is pivotal for disease diagnosis and one important class of biomarkers is constituted by proteins and peptides. This review focuses on protein and peptide analyses from biological fluids performed by capillary electrophoresis. The various strategies that have been reported to prevent difficulties due to the handling of real samples are described. Innovative techniques to overcome the complexity of the sample, to prevent the adsorption of the analytes on the inner capillary wall, and to increase the sensibility of the analysis are summarized and illustrated by different applications. To fully illustrate the contribution of CE to the analysis of biomarkers from human sample, two detailed protocols are given: the analysis from CSF of five amyloid peptide, biomarkers of the Alzheimer disease, and the analysis of sialoforms of transferrin from human serum.

Purification and aggregation of the amyloid precursor protein intracellular domain.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, ß, and γ-secretases. Sequential proteolytic cleavage of APP with ß and γ-secretases leads to the production of a small proteolytic peptide, termed Aß, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-1. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro, however it obtains stable secondary structure when bound to Fe65. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis, to modulate signaling pathways, and to regulate calcium signaling. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

Interactome of the amyloid precursor protein APP in brain reveals a protein network involved in synaptic vesicle turnover and a close association with Synaptotagmin-1.

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aß, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid ß peptide (Aß). Aß aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. Aß is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by ß- and γ-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of γ-secretase for Aß production. The interaction between APPTM and γ-secretase determines the production of different species of Aß. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP-APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D (15)N-(1)H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.

Detecting aß*56 oligomers in brain tissues.

Since its original description in 1906 by Dr Alois Alzheimer, amyloid plaques and neurofibrillary tangles have remained the hypothetical cause of Alzheimer's disease. However, plaque burden poorly predicts cognitive status in humans, which led several groups to investigate the possibility that soluble species of amyloid-beta (Aß) peptides could be playing an important pathological function in the aging brain. Through a multistep fractionation protocol, we identified a 56 kDa oligomer of Aß, termed Aß*56, the amount of which correlates with cognitive impairment. Here, we describe our biochemical approach to isolate this oligomeric Aß species in brain tissue of transgenic mouse models of AD.

[Expression and purification of a recombinant transmembrane domain amyloid precursor protein associated with Alzheimer's disease].

More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer's disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid beta-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686-726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the (1)H-(15)N-HSQC spectra, we developed APPtm(686-726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles.

Identification of novel N-terminal fragments of amyloid precursor protein in cerebrospinal fluid.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system. Two pathological hallmarks in the brain of AD patients are neurofibrillary tangles and senile plaques. The plaques consist mainly of beta-amyloid (Abeta) peptides that are produced from the amyloid precursor protein (APP), by sequential cleavage by beta- and gamma-secretase. Most previous studies have been focused on the C-terminal fragments of APP, where the Abeta sequence is localized. The purpose of this study was to search for N-terminal fragments of APP in cerebrospinal fluid (CSF) using mass spectrometry (MS). By using immunoprecipitation (IP) combined with matrix-assisted laser desorption/ionization time-of-flight MS as well as nanoflow liquid chromatography coupled to high resolution tandem MS we were able to detect and identify six novel N-terminal APP fragments [APP((18-119)), APP((18-121)), APP((18-122)), APP((18-123)), APP((18-124)) and APP((18-126))], having molecular masses of approximately 12 kDa. The presence of these APP derivatives in CSF was also verified by Western blot analysis. Two pilot studies using either IP-MS or Western blot analysis indicated slightly elevated levels of N-terminal APP fragments in CSF from AD patients compared with controls, which are in need of replications in independent and larger patient materials.

Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.

The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate.

Using a monoclonal antibody against the entire C-terminal end of human APP(695) (643-695 sequence) and a monoclonal antibody directed against human beta[1-40] amyloid peptide (betaA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of betaA. According to the nature of N- and C-terminal amino acids we identified endogenous beta-, gamma-, epsilon-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the beta-amyloid sequence at same sites as those observed in human betaA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study betaA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human betaA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.

Tissue processing prior to protein analysis and amyloid-beta quantitation.

Amyloid-containing tissue, whether from human patients or an animal model of a disease, is typically characterized by various biochemical and immunohistochemical techniques, many of which are described in detail in this volume. In this chapter, we describe a straightforward technique for the homogenization of tissue prior to these analyses. The technique is particularly well-suited for performing a large number of different biochemical analyses on a single mouse brain hemisphere. Starting with this homogenate, multiple characterizations can be done, including Western blot analysis and isolation of membrane-associated proteins, both of which are described here. Additional analyses can readily be performed on the tissue homogenate, including the ELISA quantitation of Abeta in the brain of a transgenic mouse model of beta-amyloid deposition. The ELISA technique is described in detail in the following chapter.

Crystallization and preliminary crystallographic studies of the copper-binding domain of the amyloid precursor protein of Alzheimer's disease.

Alzheimer's disease is thought to be triggered by production of the amyloid beta (Abeta) peptide through proteolytic cleavage of the amyloid precursor protein (APP). The binding of Cu2+ to the copper-binding domain (CuBD) of APP reduces the production of Abeta in cell-culture and animal studies. It is expected that structural studies of the CuBD will lead to a better understanding of how copper binding causes Abeta depletion and will define a potential drug target. The crystallization of CuBD in two different forms suitable for structure determination is reported here.

Binding of F-spondin to amyloid-beta precursor protein: a candidate amyloid-beta precursor protein ligand that modulates amyloid-beta precursor protein cleavage.

Amyloid-beta precursor protein (APP), a type I membrane protein, is physiologically processed by alpha- or beta-secretases that cleave APP N-terminal to the transmembrane region. Extracellular alpha-/beta-cleavage of APP generates a large secreted N-terminal fragment, and a smaller cellular C-terminal fragment. Subsequent gamma-secretase cleavage in the transmembrane region of the C-terminal fragment induces secretion of small extracellular peptides, including Abeta40 and Abeta42, which are instrumental in the pathogenesis of Alzheimer's disease, and intracellular release of a cytoplasmic tail fragment. Although APP resembles a cell-surface receptor, no functionally active extracellular ligand for APP that might regulate its proteolytic processing has been described. We now show that F-spondin, a secreted signaling molecule implicated in neuronal development and repair, binds to the conserved central extracellular domain of APP and inhibits beta-secretase cleavage of APP. Our data indicate that F-spondin may be an endogenous regulator of APP cleavage, and suggest that the extracellular domains of APP are potential drug targets for interfering with beta-secretase cleavage.