18 research outputs found
Single-molecule detection and characterisation of alpha-synuclein aggregates
Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders.
It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in
Parkinson’s Disease (PD), and it is widely accepted that small α-syn oligomers are the key
cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise
using traditional biochemical ensemble methods due to their high level of heterogeneity
and low abundance. Single-molecule fluorescence microscopy techniques have emerged
as a suitable approach to circumventing this problem, enabling the detection of individual
aggregates amongst monomeric protein and thus facilitating the identification, quantification,
and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation
are poorly understood. Furthermore, there remains some limitations to the singlemolecule
techniques currently available. This thesis describes the work completed to address
some of these issues.
Chapter 1 provides the contextual background for the work presented in this thesis, detailing
the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining
the single-molecule techniques used to investigate aggregate species. Chapter 2 describes
the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made
using the single-molecule techniques which were utilised and developed in this work.
One primary approach for studying species in single-molecule experiments involves directly
labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation
have previously been identified using fluorescently tagged α-syn and single-molecule Förster
resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate
formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled
α-syn to detect and characterise aggregates formed both intracellularly and in vitro
via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and
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total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and
structures of the oligomers formed. This work reveals the presence of distinct oligomeric
species in vitro and in neurons resulting from structural conversion during early aggregate
formation.
The approach taken in Chapter 3 is highly suitable for investigating aggregate formation
resulting from the addition of exogenous α-syn to samples of interest. However, such an
approach is not ideal for the detection and characterisation of endogenous aggregates due to
issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used
as an alternative approach to labelled protein; however, such dyes are non-protein-specific
and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in
Chapter 4 describes a novel single-molecule method to specifically detect and characterise
α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with
orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule
confocal microscopy. This enables the quantification and size approximation of α-syn aggregates
at picomolar concentrations, both in vitro and in biological samples.
Although the kinetics of α-syn aggregation have been studied extensively, much of our current
knowledge stems from ensemble averaging techniques which are associated with high levels
of variability and are not conducive to detecting the earliest steps in aggregate formation.
In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational
modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects
of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D
mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4.
This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new
insights into the aggregation of α-syn variants.
Overall, the work presented here provides new insights into the aggregation of α-syn via the
use and development of single-molecule techniques. The advancements made have added
to the current understanding of the molecular mechanisms of α-syn aggregation, both in
vitro and in neurons, and have also been used to develop a novel single-molecule detection
method for α-syn aggregates. The work presented in this thesis has resulted in two published
papers, ’Pathological structural conversion of alpha-synuclein at the mitochondria induces
neuronal toxicity’ in Nature Neuroscience, and ’Single-molecule two-color coincidence detection
of unlabeled alpha-synuclein aggregates’ in Angewandte Chemie International Edition.
Furthermore, the novel detection method presented here holds promise for measuring α-syn
oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates.
This may therefore be used in future studies for identifying, detecting, and studying
potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected
that the work from this thesis will be used to aid researchers towards better understanding the
mechanisms of α-syn aggregation, both in vitro and in clinical samples
Osteoclast Cytomorphometry and Scanning Electron Microscopy of Bone Eroded Surfaces During Leukemic Disorders
Tartrate resistant acid phosphatase (TRAP) is a reliable histochemical marker of osteoclasts when used on tissue sections of undecalcified bone. This paper presents an original morphometric analysis which can be done after histochemical identification of osteoclasts. These bone resorbing cells were demonstrated on undecalcified bone biopsies from control subjects and patients presenting a malignant disease of the lymphocyte B lineage. Computerized analysis of the osteoclastic population revealed that: (1) all TRAP positive cells along bone trabeculae belong to a osteoclastic population; (2) that B cell malignancies had an increased bone resorption. At the scanning electron microscopic level small resorption bays (about 10 μm in diameter) were observed either associated or separated from eroded surfaces presenting a normal appearance; TRAP staining of histological sections of undecalcified bone, coupled with morphometric studies, may help in the understanding of bone disease pathobiology
Determining the location of the α-synuclein dimer Interface using native top-down fragmentation and isotope depletion-mass spectrometry
α-Synuclein
(αSyn), a 140-residue intrinsically disordered
protein, comprises the primary proteinaceous component of pathology-associated
Lewy body inclusions in Parkinson’s disease (PD). Due to its
association with PD, αSyn is studied extensively; however, the
endogenous structure and physiological roles of this protein are yet
to be fully understood. Here, ion mobility-mass spectrometry and native
top-down electron capture dissociation fragmentation have been used
to elucidate the structural properties associated with a stable, naturally
occurring dimeric species of αSyn. This stable dimer appears
in both wild-type (WT) αSyn and the PD-associated variant A53E.
Furthermore, we integrated a novel method for generating isotopically
depleted protein into our native top-down workflow. Isotope depletion
increases signal-to-noise ratio and reduces the spectral complexity
of fragmentation data, enabling the monoisotopic peak of low abundant
fragment ions to be observed. This enables the accurate and confident
assignment of fragments unique to the αSyn dimer to be assigned
and structural information about this species to be inferred. Using
this approach, we were able to identify fragments unique to the dimer,
which demonstrates a C-terminal to C-terminal interaction between
the monomer subunits. The approach in this study holds promise for
further investigation into the structural properties of endogenous
multimeric species of αSyn
Développement d'un système d'analyse d'images numérisées pour l'étude histomorphométrique du tissu osseux
International audienc
Improved algorithms for automatic bone histomorphometry on a numerized image analysis system
International audienceQuantitative histological methods have proved to be the most effective methods in bone disease research. Faster and more accurate techniques are currently needed. We have developed a simple digitized image analysis system which allows accurate measurements of trabecular bone mass. The algorithm is based on the 'four-connected sets' mathematical theory. Given a numerized image displayed by a CCD camera, the algorithm recognizes all possible four-connected sets and provides area measurements. The first procedure automatically eliminates small, irrelevant profiles (wrinkles, cell nuclei, etc.) while larger profiles are erased interactively. The second procedure similarly erases the artefactual defects within the trabecule (artefactual cracks or empty osteocytic lacunae). The method was shown to be very accurate and time-saving