The role of p75NTR in cholinergic basal forebrain structure and function

The role of the p75 neurotrophin receptor (p75NTR) in adult cholinergic basal forebrain (cBF) neurons is unclear due to conflicting results from previous studies and to limitations of existing p75NTR-knock-out mouse models. In the present study we used a novel conditional knock-out line (ChAT-cre p75in/in) to assess the role of p75NTR in the cBF by eliminating p75NTR in choline acetyl-transferase-expressing cells. We show that the absence of p75NTR results in a lasting increase in cBF cell number, cell size, and cholinergic innervation to the cortex. Analysis of adult ChAT-cre p75in/in mice revealed that mutant animals show a similar loss of cBF neurons with age to that observed in wild-type animals, indicating that p75NTR does not play a significant role in mediating this age-related decline in cBF neuronal number. However, the increased cholinergic axonal innervation of the cortex, but not the hippocampus, corresponded to alterations in idiothetic but not allothetic navigation. These findings support a role for p75NTR-mediated regulation of cholinergic-dependent cognitive function, and suggest that the variability in previous reports of cBF neuron number may stem from limited spatial and temporal control of p75NTR expression in existing knock-out models

Measurement of uPAR and αvꞵ6 for early stage colorectal cancer diagnosis

Theoretical thesis.Includes bibliographical references.1. Introduction -- 2. Project outline -- 3. Methods -- 4. Results -- 5. Discussion.Colorectal cancer (CRC), characterised by tumours of the colon, rectum and appendix, is the 3rd leading cause of cancer-related death worldwide. The survival rate is dependent on the time of diagnosis, with early-stage detection leading to curative surgical resection. Despite this, a lack of accurate, sensitive and specific tests means that only ~30% of cases are diagnosed early enough to be cured (i.e., American Joint Committee on Cancer Stages I & II). To address this unmet clinical need, this thesis addressed developing a mass spectrometry (MS)-based assay for the detection and quantification of early-stage blood-based biomarkers of CRC. Here, we have developed targeted assays for the potential cancer biomarkers urokinase plasminogen activator receptor (μPAR) and integrin αvꞵ6. A growing portfolio of evidence implicates both proteins as important regulators of the epithelial-mesenchymal transition (EMT), a mechanism known to be critical in cancer proliferation, progression, invasion and eventual metastasis. The biggest challenge in detection and quantification of these biomarkers in plasma is their relatively low abundance (low ng/mL) compared to more highly abundant homeostatic proteins (high mg/mL). To circumvent this challenge, we developed a two-step assay that captures the proteins by affinity-based enrichment prior to quantitation by targeted multiple reaction monitoring (MRM) MS. Initial studies were aimed at determining the specificities of a range of monoclonal antibodies for their intended target. Subsequent studies using a surrogate parallel reaction monitoring (PRM)-MS established optimal peptide transitions, as well as each assay’s limits of detection. Collectively, the amalgamation of these two components (with the use of labelled peptide standards) will result in highly sensitive, specific and reproducible assays to detect and quantify μPAR and αvꞵ6 in control and diseased patient plasma. Early stage diagnosis will shift the percentage of CRCs detected towards the localized tumour stages (I and II), with consequential increased patient survival.1 online resource (74 pages : illustrations

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins

A robust method for particulate detection of a genetic tag for 3D electron microscopy

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/ gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.</p