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- Sackmann, Valerie, 1990-
(author)
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The Propagation of Neurodegenerative Diseases by Inflammation and Exosomes
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases with rates increasing along with the ageing global population. Despite best efforts, we still do not understand the etiopathogenesis of these diseases and there are no effective disease-modifying treatments. Cognitive deficiencies or motor complications that emerge during AD and PD are thought to be the result of the accumulation of misfolded, aggregate-prone proteins, such as amyloid-β (Aβ) and tau or α-synuclein (α-syn), respectively. Growing evidence suggests that prefibrillar oligomers of Aβ and α-syn (oAβ and oα-syn) are key contributors to the progression of these diseases. The progressive accumulation of these proteins leads to a gradual spread of pathology throughout interconnected brain regions, but the mechanisms by which this spreading occurs are still largely unknown.Neuroinflammation has been recognised as an important contributor to neurodegenerative disease. It is hypothesised that a pro-inflammatory environment initiated by the innate immune system, either through activation from Aβ itself or indirectly through neuronal injury signals in AD. These phenomena are thought to either cause or accelerate AD, such that an anti-inflammatory approach may be neuroprotective. In paper I, we investigated whether different inflammatory environments affected the transfer of oAβ between neuron-like cells, in addition to investigating inter- and intracellular protein changes. This study demonstrated that an anti-inflammatory environment reduces the transfer of oAβ between cells. We also provide evidence that these cells begin to take on the “phenotype” of the inflammatory milieu, while also demonstrating that the expression profile of endosomal/lysosomal and protein trafficking proteins is altered during these conditions.Small extracellular vesicles called exosomes, which are key players in cell to cell communication, have been proposed to play an influential role in spreading neurodegenerative proteins between cells. Exosomes are small membranous vesicles that are formed by the inward budding of multivesicular bodies (MVBs). These MVBs can then merge with the plasma membrane to be released into the extracellular environment as vesicles, which serve as vehicles for transferring proteins, lipids, and mRNAs between cells.The ESCRT-dependent pathway is the most understood mechanism underlying exosome biogenesis. However, exosomes can also be formed through ESCRT-independent pathways, including through the hydrolysis of sphingomyelin by neutral sphingomyelinase 2 (nSMase2), which produces ceramide. Paper II investigated whether exosomes formed through an ESCRT-independent pathway plays a significant role in the transfer of oα-syn between neuron-like cells. As oxidative stress is a common feature in PD brains, which in turn dysregulates nSMase2 activity, we also tested our model under hypoxic conditions. Inhibition of nSMase2 significantly reduced the transfer of oα-syn between cells but also resulted in decreased α-syn aggregation. Hypoxia did not influence oα-syn transfer, however, it significantly dysregulated the sphingolipid composition, which may be important for α-syn binding to exosomes and exosome communication.During AD and PD, there is a noted reduction in the effectiveness of autophagy, a process critical to cellular proteostasis. Recent studies have uncovered shared regulatory mechanisms of exosome biogenesis and autophagy, suggesting that they are closely linked. Previous findings have shown that inhibition of autophagy in AD mice mediates Aβ trafficking through altering the secretion of Aβ in MVBs. To further study this effect, we investigated the interplay between autophagy and exosome secretion using ATG7 knock-out x APPNL-F knock-in AD mice in paper III. These autophagy-deficient AD mice had a reduced extracellular Aβ plaque load, but increased intracellular Aβ, which was found to be assembled into higher-ordered assemblies. While exosomal secretion was dysregulated in these mice, the amount of Aβ packaged into the exosomes was unchanged.Lastly, one of the biggest challenges in developing effective treatments for AD is the lack of early diagnosis of living patients. As the connection between exosomes and the spread of neurodegenerative proteins is still relatively new, there remains a diagnostic potential to be explored with exosomes. Paper IV aimed to develop a new diagnostic assay to detect oAβ in exosomes isolated from human cerebrospinal fluid. Although exosomal oAβ was readily detected in some of these samples, the assay’s sensitivity requires additional optimisation before it can be further validated for the clinic.In summary, the studies presented in this thesis have furthered our understanding of how inflammation, autophagy, and exosomes contribute to the intercellular transmission of AD and PD associated proteins. We have shown that an anti-inflammatory approach may slow down the progression of AD through reducing the transfer of oAβ between cells. We also provide novel findings relating to the biogenesis of exosomes, which in turn affected the ability of exosomes to transmit neurodegenerative proteins between cells, and their association with autophagic processes. Finally, we have investigated the feasibility of exosomes as an early AD diagnostic marker. This work has helped to elucidate some of the mechanisms underlying the progression of neurodegenerative diseases, which may be useful targets for the investigation of new therapeutic avenues.
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| 2. |
- Sackmann, Christopher, 1988-
(author)
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Investigation of the intercellular transmission of α-synuclein, amyloid-β and TDP-43
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS) are disorders characterized by the progressive deposition of proteinaceous inclusions throughout the brain in a predictable manner. Each disease is described by the involvement of different misfolded and aggregated proteins (AD, amyloid-β and tau; PD, α-synuclein; ALS and FTLD, TDP-43) that spread between anatomically connected brain regions, causing cell death in previously healthy regions. Disease progresses as these aggregated proteins spread throughout the brain in a prion-like fashion. Oligomeric forms of these proteins (aggregates comprising of ≈3-30 individual proteins) are thought to be the most relevant to disease, as they are capable of prion-like propagation and can cause cellular toxicity. The work in this thesis aims to elucidate the mechanisms by which different neurodegenerative disease related proteins (amyloid-β, α-synuclein and TDP-43) are taken up and transferred between cells, and the effects exerted by these proteins on downstream cells.Paper I examined the uptake and cell to cell transmission of oligomeric α-synuclein (α-syn). Using a 3D co-culture model, we determined that α-syn (monomeric, oligomeric and fibrillar assemblies) were readily taken up and transferred between neuron-like cells, and that this transfer was mediated by an endosomal/lysosomal mechanism. It was also determined that larger α-syn assemblies (oligomers and fibrils) were found in donor and acceptor cells more frequently than monomeric α-syn, which we speculate is a due to the larger aggregates’ resistance to cellular proteases.In Paper II, we identified a novel mechanism for the uptake of oligomeric proteins, in the discovery that the gap junction channel protein connexin 32 mediates the uptake of α-syn oligomers in a preferential manner. Gap junction proteins act as a means of communication between adjacent cells, forming a transmembrane pore to facilitate the passage of small molecules. Here, we determined that connexin 32 drives the preferential uptake of oligomeric α-syn relative to monomeric and fibrillar α-syn. This system was not exclusive to α-syn however, as the preferential uptake of oligomeric amyloid-β (Aβ) was also observed. In addition to the uptake of oligomers, we observed that increased α-syn expression elicited the increased expression of connexin 32, in a positive feedback mechanism. When connexin 32 was inhibited pharmacologically or knocked out using CRISPR/Cas9, the preferential uptake of oligomers was abolished. These phenomena were also observed in oligodendrocytes (the accumulation of oligomeric α-syn in oligodendrocytes is a hallmark of Multiple Systems Atrophy), three different mouse models of α-syn overexpression, as well as in post-mortem human tissues.Paper III undertook the investigation of cell to cell transfer of TDP-43. Although it was recently confirmed that TDP-43 propagates throughout the brain in a prion-like fashion, it remains unclear how post-translational modifications of TDP-43 affect its propensity to be transferred between cells. This leaves a gap in the understanding of how TDP-43 proteinopathies progress, as post-translationally modified TDP-43 is understood to be critical to pathogenesis. To study this, we generated several TDP-43 cell lines, expressing full-length TDP-43 or C-/N-terminally truncated fragments, known contributors to TDP-43 proteinopathies. Using the 3D co-culture model, we determined that preservation of the N-terminus of TDP-43 enhanced its ability to transmit between cells, whereas an intact the C-terminus reduced transfer. Additionally, since we have previously shown that both oligomeric Aβ and α-syn are incorporated into extracellular vesicles (EVs) such as exosomes, and that these EVs can sufficiently mediate the transfer of protein oligomers to downstream cells, we investigated whether this was also true for TDP-43. We demonstrated that full-length TDP-43 and TDP-43 fragments could be found within EVs generated by these cells, but that these EVs were unable to propagate the protein to downstream cells. Instead, the transmission of TDP-43 occurs in a manner dependent upon physical proximity between cells, possibly across the synaptic cleft itself.Next, we studied the acute effects exerted by oligomeric Aβ upon healthy neurons in order to understand the earliest effects of oligomeric Aβ challenge. In Paper IV, we used iPSC-derived neurons generated from human donors expressing different amyloid-β precursor protein (APP) genes, one harbouring the familial AD-causing V717I London mutation, the other expressing WT APP. After differentiating these cells into functional neurons in vitro, the neurons were challenged with acute exposure to exogenous oligomeric Aβ and analyzed by LC-MS/MS to observe the early effects. By analyzing the proteome and phosphoproteome of these cells, we identified many proteins and phosphoproteins that were up- or down-regulated in response to oligomeric Aβ at this early timepoint. Among these changes, oligomeric Aβ caused the downregulation of TDP-43, heterogeneous nuclear ribonucleoproteins, and coatomer complex I proteins. Conversely, increases were observed in 20S proteasome subunits and vesicle associated proteins VAMP1/2. We also observed the differential phosphorylation of tau at serine 208, indicating that phosphorylation at this residue might be an important early event in tauopathy.Altogether, the work described in this thesis has provided new understanding as to how different neurodegenerative disease related proteins are taken up and transferred between cells. In doing so, we have identified some of the mechanisms by which this spreading occurs, and that the changes elicited by these toxic oligomeric proteins are rapid and widespread. By learning about these processes, we have identified novel targets that could be used in the development of disease modifying therapeutics.
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