Background: Patients with palliative-stage cancer often suffer from a variety of debilitating symptoms which have been shown to appear in clusters. It is suggested that cytokines cause many such symptoms, and elevated cytokine production has been shown to correlate with symptoms. However, symptom clusters have not been thoroughly analyzed in relation to cytokine clusters. The aim of the present study was to identify symptom clusters and cytokine clusters in Swedish cancer patients, and to investigate correlations between the identified symptom clusters and cytokine clusters. Methods: The EORTC Quality of Life Questionnaire Core 15 Palliative Care questionnaire was completed by 110 cancer patients, with blood samples taken at two time points four weeks apart. Meso scale discovery (MSD) assays were used to analyze 23 cytokines. Statistical analysis was performed using principal component analysis (PCA) of symptoms and cytokines, followed by correlation analysis of the obtained clusters. Results: Three symptom clusters were identified: (I) pain-sleep disorder, (II) gastro-intestinal-fatigue, (III) physical functioning. The cytokines were divided into three clusters that can be characterized as (I) pro-tumorigenic, (II) cell-mediated immune response and (III) proinflammatory. At the second time point, a fourth cytokine cluster was isolated (IV) immunostimulation. Correlations were found at both time points between the proinflammatory cytokine cluster and the physical functioning symptom cluster, and at the week four time point between the proinflammatory cytokine cluster and the gastro-intestinal-fatigue symptom cluster. Conclusions: We show a correlation between symptom clusters and the proinflammatory cytokine cluster. Proinflammatory cytokines are known to cause symptoms that resemble palliative cancer symptoms. Increased knowledge of biochemical processes and their effect on patients’ wellbeing may give clues for counteracting symptoms that affect quality of life (QOL) in palliative cancer care. © Annals of Palliative Medicine. All rights reserved.

The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the proteins transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology.

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 APP^NL-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.