1,721,053 research outputs found
Investigating the structure of alpha-synuclein using mass spectrometry
Full text linkThe pathological hallmark of Parkinson’s disease (PD) are Lewy bodies (LBs),
insoluble inclusions observed in dopaminergic neurons in the brains of PD patients.
The main protein component of LBs is alpha-synuclein (αSyn), a 140-residue
intrinsically disordered protein. Around 10% of PD cases are associated with genetic
mutations, including single-point variants of αSyn; and under physiological
conditions, the protein carries a constitutive N-terminal acetylation modification.
Thus, the structural and functional properties associated with αSyn are vitally
important to investigate in order to further understanding of how this protein
contributes to disease.
Here, we report the first full biophysical characterisation and cross-comparison
of wild-type (WT) αSyn and a panel of PD-associated variants, using circular dichroism
spectroscopy, fluorescence aggregation assays, native mass spectrometry, and ion
mobility-mass spectrometry (IM-MS). We uncover that the different variants occupy
different conformational spaces in the gas phase, and that the monomeric proteins
do not exhibit a completely unfolded structure, as expected for a disordered protein.
The N-terminal acetylated variants of αSyn are a more physiologically relevant
model, with the constitutive modification being important for the formation of a
transient N-terminal α-helix which mediates the binding of αSyn to various cellular
lipid membranes. Here, we studied the effect of this modification on the structure
and function of the panel of αSyn variants.
The native state of αSyn is highly disputed, with several reports proposing the
existence of naturally occurring multimers of the protein that may be involved in the
physiological role of αSyn. However, these species have not been studied extensively
and their role is not fully understood. Here, IM-MS and native top-down
fragmentation using electron capture dissociation were used to elucidate the
structural properties associated with a stable dimeric species of αSyn. In addition, we
integrated a novel method for generating isotopically depleted protein into our
native top-down workflow. Isotope depletion increases signal to noise ratio and/or
reduces the spectral complexity of fragmentation data, enabling the monoisotopic
peak of low abundant fragment ions to be observed. Using this new workflow, we
were able to infer structural information about this previously unreported αSyn
dimer interface.
Overall, this body of work aims to highlight native mass spectrometry as an
important tool for investigating challenging structural biology problems, such as
intrinsically disordered and aggregating proteins. This work also represents a
comprehensive structural study of physiologically relevant WT αSyn and PDassociated
variants in the gas phase. We showed that the N-terminal acetylation of
αSyn and various PD-associated variants alters all aspects of structure and function
of the protein, highlighting the need for physiologically relevant modifications to be
used in in vitro studies. We also provide conclusive evidence for a C-C terminal
interaction between the monomer units forming the stable dimeric species of αSyn,
presenting important structural data on endogenous αSyn multimers
Derivation of enkephalinergic medium spiny neurons from mouse embryonic stem cells
Full text linkMedium spiny neurons (MSNs) play an important role in locomotion.
Counterbalance between two MSN subtypes, enkephalin-positive and substance P-positive
MSNs, is crucial for maintaining normal movement. Preferential
degeneration of enkephalinergic MSNs in early stage Huntington’s disease (HD)
contributes to abnormal involuntary movement called chorea. The reasons for this
selective vulnerability are unknown. In vitro differentiation of pluripotent stem cells
(PSCs) to neuronal cells is considered a potential approach for modelling
neurodegenerative disorders including HD. Generation of PSC-derived
enkephalinergic MSNs would be an ideal tool for dissecting their preferential
degeneration. However, an enkephalinergic phenotype has never been reported in
PSC-derived MSNs. We, therefore, have generated a mouse embryonic stem cell
(mESC) reporter line that expresses enhanced yellow fluorescent protein (EYFP)
when the cells are committed to an enkephalinergic fate. Characterisation of this
mESC line via chimaera generation showed that all EYFP-positive cells were also
enkephalin-positive. We have then optimised an enkephalinergic neuronal
differentiation protocol using this ESC line. Interestingly, we found that a
combination of Wnt inhibitor Dickkopf-related protein 1 (DKK1), sonic hedgehog
(Shh) and brain-derived neurotrophic factor (BDNF), commonly used in addition to
basal medium for deriving MSNs from PSCs, had a detrimental effect on enkephalin
expression. Absence of these three factors, surprisingly, did not reduce the potential
of ESCs to become MSNs nor did it affect the electrophysiological properties of
ESC-derived MSNs. Further investigation revealed that Pre-pro-enkephalin is down-regulated
in the presence of exogenous DKK1 and/or Shh but not in the presence of
BDNF. We, therefore, propose that addition of exogenous DKK1 and Shh is
unfavourable to derive enkephalinergic MSNs from mouse ESCs. These findings
could be used to derive enkephalinergic MSNs in vitro allowing the disease in a dish
approach for HD modelling
Characterization of the developing haematopoietic stem cell niche using a novel immortalization system
Full text linkEmbryonic haematopoiesis is a complex process under intensive research. Murine
definitive Haematopoietic Stem Cells (HSCs) originates from the
Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that
definitive HSCs arise from endothelial lining of dorsal aorta.
However, detail of
HSC specification in the developing embryo remains elusive. One way to
deciphering events occurred during HSC specification is to derive cell lines from the
developing HSC niche. Previous work by Oostendorp et al. showed the AGM and
fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al.
2002). In this study, I established a more robust immortalization system using
normal SV40 large T antigen delivered via Neon™ electroporation system. The new
immortalization system achieved direct immortalization without going through crisis.
And it is compatible with small number of primary cells dissected from different
haematopoietic niches. With my new system, multiple cell lines from different
haematopoietic sites at different developmental points are derived. Moreover, some
of these lines demonstrated ability to mature precursors from E9.5 embryo
(pro-HSCs) to definitive HSC without help of growth factors. This result is better
compared to OP9 stromal lines. Such data proved usefulness of using stromal cell
lines to study haematopoietic specification
Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated mutation of the SNCA gene
Full text linkAn experimental treatment for Parkinson’s disease (PD) involved the transplantation of fetal midbrain tissue, a source of midbrain dopaminergic (mDA) progenitors, into the striatum of patients to restore dopaminergic innervation. Although clinical benefits were experienced by some patients, this heterogeneous and scarce source of tissue is not sustainable. Recently, mDA progenitors differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are comparably potent and efficient as fetal midbrain tissue in rescuing dopaminergic deficits in PD animal models and clinical trials using this cell product are progressing. However, the hESC/hiPSC-derived mDA grafts are still susceptible to the development of Lewy body pathology, as found in clinical trials using fetal tissues. The clinical benefits of the fetal grafts reduced in correlation with the accumulation of Lewy body pathology, therefore, a pathology-resistant graft would be longer-lasting and beneficial to patients. For in vitro modelling of Lewy body pathology, which is an inclusion pathology mainly consist of misfolded α-Synuclein (α-Syn) aggregates, I treated hESC-derived mDA neurons with α-Syn pre-formed fibrils (PFFs). PFFs recruit endogenous α-Syn to form Lewy body-like aggregates which are positive for phospho-serine 129 α-Syn (pS129-αSyn), ubiquitin and p62. The PFF models also recapitulate other aspects of PD, such as synaptic and mitochondrial dysfunction, neuroinflammation and neurodegeneration. Since endogenous α-Syn is essential for the development of aggregates, I attempted to produce pathology-resistant neurons by knocking out α-Syn (SNCA) in hESCs with CRISPR/Cas9n and subsequently differentiating the SNCA+/– and SNCA–/– hESCs into mDA neurons. As α-Syn might play physiological roles which are not yet fully elucidated, I created a single amino acid (S87E) mutation in α-Syn, aiming to reduce α-Syn aggregation without disruption of α-Syn physiological functions. Half of the resulting CRISPR-engineered SNCA+/–, SNCA–/– and SNCAS87E/S87E hESC clones exhibited normal genomic integrity, free from detectable copy number variations (CNVs), large copy-neutral loss of heterozygosity (CN-LOH), off-target events and integration of targeting plasmids. Subsequently, mDA neurons were differentiated from SNCA+/–, SNCA–/– and SNCAS87E/S87E hESCs and they highly resembled mDA neurons derived from WT parental hESCs based on marker analysis and RNAseq. This data suggested that the α-Syn mutations, as well as the selection and cloning process, did not impair mDA differentiation. Synapse formation, spontaneous activities and dopamine secretion were readily observed in mDA neurons of all tested genotypes. The WT mDA neurons treated with PFFs recapitulated pS129-αSyn pathology, but did not result in detectable cell death or significant impairment of synapse formation, mitochondrial morphology or spontaneous neuronal activities within the timeframe of the current study. SNCA+/–, SNCA–/– and SNCAS87E/S87E mDA neurons treated with PFFs revealed that SNCA+/– exhibited significantly less, while SNCA–/– showed no pS129-αSyn pathology and SNCAS87E/S87E exhibited a reduced level of pathology compared to WT mDA neurons. The PFF-treated hESC-derived mDA neuron model established in this study could be used as an effective platform for drug screening. In addition, SNCA+/– and SNCA–/– hESC-derived cells could be valuable cell models for studying the physiological role of α-Syn. On the condition of satisfactory validation in animal models, SNCA+/– and SNCA–/– hESC-derived mDA progenitors would have significant potential in cell replacement therapy for PD
Investigating the protein targets of the neuroprotective E3 ligase, CHIP
Full text linkIdentifying early defects in protein homeostasis (“proteostasis”) in neurodegeneration
could shed light into disease-promoting events and provide promising therapeutic
targets for disease modification. A key player in the regulation of proteostasis is the
dual function chaperone and E3 ligase C-terminus of Hsc-70 interacting protein
(CHIP). Its role in neurodegenerative diseases, including dementia with Lewy bodies,
and the severe progeria and proteotoxic phenotype seen in CHIP KO mice support its
neuroprotective effects, but the underpinning molecular mechanisms remain largely
unknown and understudied. This project aimed to identify the protein targets of CHIP
in a neuronal cell model of disease.
I knocked out CHIP expression using CRISPR/Cas9 technology from inducedpluripotent
stem cells (iPSC) derived from a synucleinopathy patient with dementia
and differentiated them into cortical neurons. A label-free quantitative mass
spectrometry to analyse CHIP-dependent changes was conducted and the proteome
defined. This comparative proteomic analysis revealed that of all the proteins
identified and significantly changed between CHIP KO and WT lines, only 35 proteins
could be (directly or indirectly) regulated by CHIP. This supports the emerging
hypothesis that CHIP can be chaperone-independent docking-dependent E3 ligase,
having tight specificity for substrates, in this model of synucleinopathy with mild
proteotoxic stress.
Annexin and other calcium-binding proteins, in particular Annexin A2 and its
interacting protein S100-A11, were the most over-represented proteins in the CHIP
KO cortical neurons. From this comprehensive target discovery investigation, I have
validated Annexin A2 increases in CHIP KO cells and have detected an endogenous
Annexin A2:CHIP interaction, both in vitro and in situ, in different CHIP cell models.
Moreover, CHIP-dependent ubiquitination of Annexin A2 was also identified, both in
vitro and in situ. Single-chain antibodies against CHIP have been engineered to
modulate its activity. By collapsing the higher molecular weight structures of CHIP,
CHIP-dependent ubiquitination of Annexin A2, but not other substrates, is enhanced.
Annexins are a conserved family of calcium-regulated phospholipid-binding proteins
that are required for membrane repair and maintenance of membrane homeostasis.
Given these functions, several types of membrane damage assays were conducted
and suggested that CHIP KO cells are more sensitive to damage, despite retaining
the ability to repair. This impairment in membrane resilience was also seen in CHIP
KO cells expressing a E3 ligase-dead CHIP mutant, but the phenotype was partly
rescued in the cell lines expressing a chaperone-dead CHIP mutant or wild-type
CHIP. Compromised Annexin A2:S100A11 interactions (important for the repair
complex) and a different lipidomic profile between CHIP WT and KO cells could
contribute to this phenotype.
Although there are reports of annexins being overexpressed in some
neurodegenerative diseases, there have been no follow-up studies deciphering the
molecular mechanisms of annexins within neurons. I have identified Annexin A2 as a
substrate for CHIP and revealed other novel calcium-regulated membrane-binding
CHIP targets. Thus, CHIP is likely to play a role in regulating membrane protein
homeostasis and maintaining membrane integrity, which may help to explain the
neuroprotective actions of CHIP. This is of relevance within the emerging field of
impaired membrane integrity in the context of neurodegeneration
Smad2/3 potentiate cell identity conversions with master transcription factors
Full text linkThe exogenous expression of master transcription factors (TFs) to drive cell identity
changes is an exciting and powerful approach to cell and tissue engineering. Yet, the
generation of desired cell types is often plagued by inefficiency and inability to produce
mature cell types. Through investigations of the molecular mechanisms of induced
pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively
active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could
dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3
interacted with both co-activators and reprogramming factors, bridging their interaction
during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in
different cell types, I tested the conversions of B cells to macrophages, myoblasts to
adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was
markedly enhanced when the master TFs were co-expressed with Smad3CA. These
results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated
cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor
that potentiates the generation of diverse cell types with profound efficiency and
maturity
Modelling synucleinopathies with human neurons derived from embryonic stem cells over-expressing α-Synuclein
Full text linkα-Synuclein (αSyn) is a small intrinsically disordered protein that drives the progression of a
group of neurological disorders known of synucleinopathies, including Parkinson's disease,
dementia with Lewy bodies and multiple system atrophy. Increased expression of αSyn due
to gene duplication or triplication causes familial forms of these diseases, of which the
severity is positively correlated with the gene copy number. Despite extensive efforts using
various models, the precise mechanisms of αSyn toxicity in neurons have not been
elucidated. This could be partly due to biological differences between the models and
authentic human neurons.
In an attempt to model synucleinopathies with human neurons, I have established a
collection of transgenic human embryonic stem cell (hESC) lines over-expressing αSyn. I
first showed that elevated αSyn expression does not affect hESC proliferation and their
differentiation potential towards neurons. Then I identified transgenic hESC lines that
maintained high αSyn expression in differentiated neurons and compared the rate of reactive
oxygen species (ROS) production in high versus normal αSyn expressing cortical neuronal
cultures. I observed a significantly elevated level of ROS production in αSyn over-expressing
neurons in less mature neurons; however, there was no difference observed in
more mature neurons. The possible reasons that lead to this difference are discussed.
This is the first report of stable αSyn overexpressing hESC lines, which can provide an
unlimited source of human neurons for studying the mechanism underlying neuronal cell
death in synucleinopathies, which in turn could lead to the development of potential
therapeutics
Molecular mechanisms of the Ca2+/Calmodulin-mediated control of actin cytoskeleton-driven neuromorphogenesis processes mediated by the actin nucleator JMY
Full text linkLocal formation of actin filaments powers neuronal morphology development and plasticity. Yet, responsible actin nucleators and their linkage to calcium transients largely remained elusive. Gain-offunction and loss-of-function studies unravel that JMY (Junction-mediating and regulatory protein) – a WH2 domain-based actin nucleator – promotes dendritic arborization and extension. This crucial function reflects JMY’s ability to promote actin filament formation, as it depended on JMY’s Arp2/3 complex coupling, Arp2/3 complex activity and presence and functionality of JMY’s WH2 domains. Importantly, WH2 domain #1 showed the tightest actin association and was regulated by the calcium sensor protein calmodulin in a reversible manner. Deletion of the calmodulin binding site or impairing actin binding of WH2 domain #1 rendered JMY dysfunctional. In line, JMY-mediated dendritic arborization relied on proper Ca2+/calmodulin signaling. Together, these results strongly suggests that Ca2+/calmodulin-control represents a thus far underrecognized, general principle for regulating actin nucleation underlying neuromorphogenesis indispensable for proper neuronal network formation
Apolipoprotein E and propagation of pathological tau in Alzheimer’s Disease
Full text linkAlzheimer’s Disease (AD) is characterised by accumulation of amyloid-b (Ab) in
plaques and hyperphosphorylated tau in neurofibrillary tangles. This occurs
alongside neuroinflammation and neurodegeneration. Clinically, AD is typified by
cognitive decline. Pathological tau propagates through the AD brain in a defined
manner, with indications this occurs trans-synaptically. Spread of pathological
tau correlates with synapse loss and cognitive decline. Neuroinflammation,
contributes to AD pathogenesis and is mediated by glia. Apolipoprotein E (APOE)
genotype is the strongest genetic risk factor for the late-onset AD, with APOE4
increasing risk and APOE2 conferring protection. The exact mechanisms by
which APOE modulates AD risk remain to be comprehensively discerned.
APOE influences Ab pathology, but distinct roles in neurodegeneration and Ab-independent mechanisms are less clear. A systematic literature review was
performed to assess APOE effects on neurodegeneration, neuroinflammation
and spread of pathological proteins in AD. We identified isoform-specific roles for
APOE in neurodegeneration and neuroinflammation. APOE likely mediated some
interplay between these processes. We also identified the need for multiple
approaches to understand the complex and multifaceted role of APOE in AD
pathogenesis. No studies directly investigated whether APOE genotype impacts
tau propagation.
The trans-synaptic hypothesis of tau spread requires pathological tau to be
located at synapses in human brain. This thesis characterised the synaptic
localisation of misfolded tau in control and AD human post-mortem brain and
analysed this in the context of APOE genotype. Misfolded tau was present at
synaptic pairs and was increased in AD brain. Thus, trans-synaptic spread is
credible in human AD. Asymmetric distribution of tau across synapses suggested
an anterograde mode of transmission, while presence at both synaptic terminals
suggested tau propagates across intact synapses. APOE4 did not affect synapse
loss but did impact synapse volume. Control APOE4 carriers exhibited similar
phenotypes to AD cases, suggesting APOE4 effects on synaptic tau, and
potential downstream effects on trans-synaptic spread, occur early in AD.
Astrocytes have been implicated in synapse loss and tau propagation in model
systems. Whether this translates to human brain is unclear. APOE is produced
by glia and isoform-specific effects on neuroinflammation have been identified.
Thus, APOE genotype could influence glial contributions to synapse loss and
spread of tau in AD. Astrocytes in human post-mortem brain colocalised with
presynaptic material, phosphorylated tau and tau-containing presynapses.
Greater colocalisation was observed in AD brain and in APOE4 carriers. Thus, in
AD, presynapses and pathological tau appeared more prone to astrocytic
internalisation, possibly influencing synapse loss and tau spread. APOE4 mainly
increased colocalisation in control cases, suggesting APOE effects occur early in
AD pathogenesis.
These studies suggested APOE effects on AD pathogenesis occur early in
disease, before symptom onset. To directly investigate whether APOE genotype
impacts tau propagation, mice expressing human APOE isoforms or no APOE
were used. Mutant human tau was expressed in the entorhinal cortex by viral-mediated gene delivery and tau propagation quantified. Human tau spread locally
and through hippocampal circuits, supporting the trans-synaptic hypothesis of
tau propagation. APOE genotype did not influence the spread of pathological
tau, although low statistical power impeded robust conclusions being drawn.
The findings presented in this thesis demonstrate that trans-synaptic tau spread
occurs in a mouse model and is credible in the context of human AD. Moreover,
astrocytes might impact tau spread and synapse loss by internalising
pathological tau and synapses. APOE4 worsened these phenotypes, particularly
in controls, suggesting APOE effects might be particularly relevant early in
disease pathogenesis. However, this was not found to be evident in a mouse
model of tau propagation. Understanding isoform-specific effects of APOE on
tau pathology and glial processes will be instrumental in furthering our
understanding of disease mechanisms that could be therapeutically targeted
Endogenous state of α-synuclein protein alters susceptibility to Lewy-like pathology
Full text linkBackground and aims: Two human mutations affecting the α-synuclein protein confer the most clinical severity in Parkinson’s disease; endogenous αSyn over-expression due to gene multiplication and αSynG51D due to a missense mutation. The main hypotheses include 1) αSyn over-expression may affect cortical neuronal differentiation, 2) αSyn over-expression influences vulnerability to Lewy-like pathology in human cortical neurons, and 3) the SNCAG51D mutation alters susceptibility to Lewy-like pathology in a novel rat model of PD.Methods: Transgenic human embryonic stem cells (hESCs) over-expressing αSyn were differentiated into cortical neurons and analysed for gene expression. Cortical neurons were challenged with recombinant human wild-type αSyn monomers or pre-formed fibrils (PFFs) for 1 week. Subsequent analysis for phosphorylated αSyn (pS129-αSyn), an early disease marker, was performed two to three weeks later. In the second model, novel CRISPR-edited SNCAG51D/G51D rats and SNCA+/+ control rats received unilateral intracortical injections of αSyn PFFs, and pS129-αSyn structures were quantified at six months.Results: Transcriptomic analysis of hESCs and immature cortical neurons, as well as mature cortical neuron counts, showed αSyn over-expression did not impair cortical neurogenesis. Mature cortical neurons with high endogenous αSyn that were challenged with αSyn PFFs, but not αSyn monomers or unseeded neurons, readily formed pS129-αSyn inclusions (p < 0.001). In the SNCAG51D/G51D rats, αSyn PFF-injection led to defined, mature-appearing, Lewy-like (pS129-αSyn) inclusions with a predilection for the striatum in mutant rats, although not statistically significant. PFF-injected SNCA+/+ rats had diffuse pS129-αSyn pathology in similar interconnected regions to PFF-injected mutant rats. Conclusion: αSyn over-expression in hESCs did not impair differentiation into cortical neurons, which helps to clarify conflicting data in the literature on the role of αSyn in neurogenesis. Secondly, exposure to αSyn PFFs in human cortical neurons with high endogenous αSyn expression produced significantly more pS129-αSyn inclusions than neurons with wild-type levels of αSyn expression. Finally, the SNCAG51D/G51D rats developed more defined Lewy-like pathology in PD-vulnerable regions than controls after PFF-injection. These two accelerated disease models may provide further mechanistic insight in PD
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