Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aβ) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aβ or small soluble Aβ oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aβ, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.Wellcome Trust Programme Grant 094425/Z/10/

The role of the prion-like protein SOD1 and macropinocytosis in the propagation of disease in ALS; an infectious idea

With the onset of the rapidly increasing population, the impact of age related neurodegenerative diseases including Amyotrophic lateral sclerosis, Alzheimer’s disease, Creutzfeldt-Jakob disease, Parkinson’s disease, Huntington’s disease and frontotemporal dementia is becoming a predominant health and economic concern. Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular degenerative disease that currently has no effective treatment or therapeutics. ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The mechanisms underlying neurodegeneration of motor neurons and disease progression are currently unknown; however, current evidence implicates a range of cellular mechanisms. These mechanisms include, deficient protein quality control, aberrant RNA metabolism, oxidative stress, endoplasmic reticulum stress, glutamate excitotoxicity, mitochondrial dysfunction, fragmentation of the Golgi apparatus, activated glia, axonal transport defects and neuroinflammation. These dysfunctional cellular pathways may be associated with the protein aggregates that are hallmarks of ALS pathology. However, the dysfunction in several cellular processes does not explain the spreading of pathology, here the aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent accumulation and deposition in motor neurons may contribute. Given this hypothesis, the work presented in this thesis aimed to further examine the role of SOD1 and TDP-43 in the propagation of neurodegeneration in ALS, and investigate whether the proteins exhibit prion-like properties. The term “Prion-like” as used here refers to the misfolding and aggregation of a disease specific protein that subsequently escapes the cellular environment and seeds aggregation in a naïve cell. The main aims of this thesis were to: investigate the mechanisms underpinning the uptake of SOD1 aggregates into murine NSC-34 cells (Chapter 2); Examine the subsequent release of SOD1 into the cytosol, detect released extracellular SOD1, and observe for secreted SOD1 internalisation into NSC‐34 motor neurons, then identify and quantify seeding activity in recipient cells expressing SOD1 and characterise this interaction using a novel flow cytometry method to quantify protein aggregation; Flow cytometric characterisation of inclusions and trafficking (FloIT) (Chapter 3); determine whether exogenous recombinant SOD1 protein aggregates can induce and/or contribute to TDP-43 pathology (Chapter 4); determine if SOD1 aggregates can enter humanised models of motor neurons via the same mechanism of action using both iPSC derived motor neurons and primary neurons (Chapter 5)

The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases

With the onset of the rapidly aging population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer\u27s disease, Creutzfeldt-Jakob disease (CJD), Parkinson\u27s disease, Huntington\u27s disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets

Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology

Hypochlorite-induced oxidation promotes aggregation and reduces toxicity of amyloid beta 1-42

Exacerbated hypochlorite (OCl−) production is linked to neurodegenerative processes, but there is growing evidence that lower levels of hypochlorite activity are important to protein homeostasis. In this study we characterise the effects of hypochlorite on the aggregation and toxicity of amyloid beta peptide 1–42 (Aβ1-42), a major component of amyloid plaques that form in the brain in Alzheimer's disease. Our results demonstrate that treatment with hypochlorite promotes the formation of Aβ1-42 assemblies ≥100 kDa that have reduced surface exposed hydrophobicity compared to the untreated peptide. This effect is the result of the oxidation of Aβ1-42 at a single site as determined by mass spectrometry analysis. Although treatment with hypochlorite promotes the aggregation of Aβ1-42, the solubility of the peptide is enhanced and amyloid fibril formation is inhibited as assessed by filter trap assay, thioflavin T assay and transmission electron microscopy. The results of in vitro assays using SH-SY5Y neuroblastoma cells show that pre-treatment of Aβ1-42 with a sub-stoichiometric amount of hypochlorite substantially reduces its toxicity. The results of flow cytometry analysis and internalisation assays indicate that hypochlorite-induced modification of Aβ1-42 reduces its toxicity via at least two-distinct mechanism, reducing the total binding of Aβ1-42 to the surface of cells and facilitating the cell surface clearance of Aβ1-42 to lysosomes. Our data is consistent with a model in which tightly regulated production of hypochlorite in the brain is protective against Aβ-induced toxicity

Extracellular wildtype and mutant SOD1 induces ER-Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER-Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER-Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER-Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER-Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER-Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS

Corrigendum to ‘Hypochlorite-induced oxidation promotes aggregation and reduces toxicity of amyloid beta 1–42’ [Redox Biol. 63 (2023) 102736] (Redox Biology (2023) 63, (S2213231723001374), (10.1016/j.redox.2023.102736))

The authors regret to inform you that there were some typographical errors that the authors found only after the proof review process. We would like to these to be corrected as a corrigendum. Please see the corrections below: 1. Page 5/10: “Aβ1-42 compared to untreated Aβ1-42 (Fig.6Bi and ii). Under the condi” -\u3eFig. 6 Bi), need to be hyperlinked to the Figure2. Page 6/10: “Hoechst 33452 (blue)” – please rewrite to Hoechst 33342, Fig 6. Line 6 and 83. Page 8/10, Line 8 (right side): time (t1/2) – change the t to normal size, it is currently in subscript form. It should look like this “t1/2″4. Page 8/10, 4.12. Cell culture: please delete the duplicated sentence, “All cell culture reagents and media were obtained from GE Healthcare”.5. Page 10/10, Reference [40] in black font. Please make this the same as other references.The authors would like to apologise for any inconvenience caused

SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation

Background Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated. Results We demonstrate in this paper that SOD1 aggregates interact with the cell surface triggering activation of Rac1 and subsequent membrane ruffling permitting aggregate uptake via stimulated macropinocytosis. In addition, other protein aggregates, including those associated with neurodegenerative diseases (TDP-43, Htt ex1 46Q, α-synuclein) also trigger membrane ruffling to gain entry into the cell. Aggregates are able to rupture unstructured macropinosomes to enter the cytosol allowing propagation of aggregation to proceed. Conclusion Thus, we conclude that in addition to basic proteostasis mechanisms, pathways involved in the activation of macropinocytosis are key determinants in the spread of pathology in these misfolding diseases

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