Advanced glycation end products modulate amyloidogenic APP processing and Tau phosphorylation: a mechanistic link between glycation and the development of Alzheimer’s disease

Advanced glycation end products (AGEs) are implicated in the pathology of Alzheimer's disease (AD), as they induce neurodegeneration following interaction with the receptor for AGE (RAGE). This study aimed to establish a mechanistic link between AGE-RAGE signaling and AD pathology. AGE-induced changes in the neuro2a proteome were monitored by SWATH-MS. Western blotting and cell-based reporter assays were used to investigate AGE-RAGE regulated APP processing and tau phosphorylation in primary cortical neurons. Selected protein expression was validated in brain samples affected by AD. The AGE-RAGE axis altered proteome included increased expression of cathepsin B and asparagine endopeptidase (AEP), which mediated an increase in Aβ 1-42 formation and tau phosphorylation, respectively. Elevated cathepsin B, AEP, RAGE, and pTau levels were found in human AD brain, coincident with enhanced AGEs. This study demonstrates that the AGE-RAGE axis regulates Aβ 1-42 formation and tau phosphorylation via increased cathepsin B and AEP, providing a new molecular link between AGEs and AD pathology. </p

Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

AbstractCysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy

Molecular mechanisms of cellular dysfunction caused by a tauopathy-associated fragment

Tauopathies are a group of neurodegenerative diseases characterised by cognitive and motor dysfunction, and pathological aggregates of abnormally phosphorylated and cleaved tau, a microtubule associated protein. Whilst the cause of these pathological changes is unknown, it is likely to be a combination of toxic gain-of-function acquired by aggregated tau, along with a loss of normal function of tau. This laboratory previously identified a form of cleaved tau (Tau35) in post-mortem human tauopathy brain and has developed animal and cell models expressing Tau35 to investigate molecular mechanism underlying tauopathy. These models include a transgenic mouse expressing Tau35 in low amounts, and Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human tau (CHO-FL). This thesis aims to determine how Tau35 affects molecular mechanisms involved in tau metabolism, including the unfolded protein response (UPR) and autophagic/lysosomal degradation. In aged Tau35 mice, there was evidence of a toxic gain of function, including increased tau phosphorylation at disease-relevant epitopes in multiple brain regions. In addition, Tau35 mice exhibit activation of the UPR in specific brain regions. In CHO-Tau35 cells, a significant reduction in basal autophagy was identified, which was not apparent in either untransfected CHO cells or in CHO-FL cells expressing full-length tau, suggesting that Tau35 expression results in defective autophagy. Under conditions that stimulate autophagy, CHO-Tau35 cells exhibited a reduced capacity to activate mTOR-mediated autophagy and reduced autophagic flux. Conversely, expression of Tau35 promoted activation of autophagy through an mTOR-independent mechanism. Furthermore, inhibition of glycogen synthase kinase-3 rescued the defective autophagy in CHO-Tau35 cells. Notably, CHO-Tau35 cells exhibited a reduction in the number of lysosomes, as well as defective lysosomal functionality and a deficit in lysosome biogenesis compared to CHO-FL and untransfected CHO cells. Taken together, these findings suggest novel mechanisms through which the presence of Tau35, a tau fragment associated with the development of human tauopathy, exerts a detrimental effect on cells and provides insights into the significance of protein degradation systems in the development of tauopathy

In-Vivo Detection of Cathepsin-D in Alzheimer’s Disease

Alzheimer’s disease is a neurodegenerative disease with no early diagnosis. Neuronal dysfunction is an early indication of Alzheimer’s disease which we can measure by examining the lysosomal protein Cathepsin-D. Molecular imaging of Cathepsin-D using Magnetic Resonance Imaging (MRI) contrast agents could enhance visualization of disease and provide contrast within tissues and cells that are morphologically similar but physiologically distinct. The purpose of our work was to evaluate a novel MRI/fluorescent contrast agent designed to detect Cathepsin-D in early Alzheimer’s disease. In-vitro MRI and fluorescent sensitivity were characterized in addition to cellular uptake in cells over-expressing Cathepsin-D. Cortical and hippocampal uptake was evaluated following in-vivo injection of contrast agent in mice. The contrast agent exhibited differences in retention and uptake between control and transgenic Alzheimer’s mice. The results demonstrate the potential utility of the contrast agent for in-vivo identification of cathepsin-D upregulation and will continue to be further evaluated and refined in future studies

Structural and Functional Determinants of γ-Secretase, an Intramembrane Protease Implicated in Alzheimer’s Disease

Alzheimer’s disease is the most common form of neurodegenerative diseases in humans, characterized by the progressive accumulation and aggregation of amyloid-β peptides (Aβ) in brain regions subserving memory and cognition. These 39-43 amino acids long peptides are generated by the sequential proteolytic cleavages of the amyloid-β precursor protein (APP) by β- and γ-secretases, with the latter being the founding member of a new class of intramembrane-cleaving proteases (I-CliPs) characterized by their intramembranous catalytic residues hydrolyzing the peptide bonds within the transmembrane regions of their respective substrates. These proteases include the S2P family of metalloproteases, the Rhomboid family of serine proteases, and two aspartyl proteases: the signal peptide peptidase (SPP) and γ-secretase. In sharp contrast to Rhomboid and SPP that function as a single component, γ-secretase is a multi-component protease with complex assembly, maturation and activation processes. Recently, two low-resolution three-dimensional structures of γ-secretase and three high-resolution structures of the GlpG rhomboid protease have been obtained almost simultaneously by different laboratories. Although these proteases are unrelated by sequence or evolution, they seem to share common functional and structural mechanisms explaining how they catalyze intramembrane proteolysis. Indeed, a water-containing chamber in the catalytic cores of both γ-secretase and GlpG rhomboid provides the hydrophilic environment required for proteolysis and a lateral gating mechanism controls substrate access to the active site. The studies that have identified and characterized the structural determinants critical for the assembly and activity of the γ-secretase complex are reviewed here

Yeast growth selection system for the identification of cell-active inhibitors of [beta]-secretase

The production and deposition of the cytotoxic Aβ peptide is a central event in the pathogenesis of Alzheimer’s disease (AD). Aβ is excised from the amyloid precursor protein (APP) through sequential actions of the β-secretase, which cleaves at the socalled β-site, and the γ-secretase, which cleaves at the so-called γ-site of APP. Inhibition the β-secretase BACE1 is a promising approach for AD therapy, but the search for small molecule inhibitors has proven to be challenging. In this thesis I describe a novel screening assay to identify cell-active BACE1 inhibitors by a positive yeast growth selection system, which combines the practicability of in vitro assays with the advantages of a cell-based assay. Analogue to the situation in mammalian cells, the β-site cleavage reaction was reconstituted in the secretory pathway of yeast cells using membrane-bound BACE1 and a membrane-bound APP-derived substrate. In contrast to conventional mammalian cell-based assays, false positive compounds can be rapidly excluded by the use of simple specificity controls that mimic the readout in the absence of BACE1. Furthermore, there is counter-selection for toxic compounds due to the positive growth readout upon inhibition of BACE1. The system was initially validated with two bona fide BACE1 inhibitors that stimulated the growth of BACE1-expressing cultures in a concentration-dependent manner, whereas the growth of control cultures remained unaffected in the presence of these inhibitors. In order to identify novel BACE1 inhibitors and to further validate the system, we screened a library of 15’000 small molecules. This screening revealed six compounds, which significantly reduced the secretion of Aβ from a human cell line overexpressing APP