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Defects of lysosomal morphology in the presence of β proteins. (A) Fluorescence images of DIV 6+1 primary cortical neurons transfected with mCherry (top), β4-mCherry (middle), or β23-mCherry (bottom) and incubated with LysoTracker Green. White dashed lines show the contours of the neurons. Arrowheads point to β protein aggregates. Yellow circles outline lysosomes. (B) Distribution of the lysosomal size in control and β protein-expressing neurons (mCherry, n = 2,148 lysosomes from 36 cells; β4-mCherry, n = 1,595 lysosomes from 44 cells; β23-mCherry, n = 1,838 lysosomes from 46 cells; from four independent experiments; two-tailed Mann-Whitney test). (C) Box plot showing the number of lysosomes per neuron (mCherry, n = 36 cells; β4-mCherry, n = 44 cells; β23-mCherry, n = 46 cells; from four independent experiments; two-tailed Mann-Whitney test). (D) Quantification of Person's correlation coefficient between the mCherry and LysoTracker signal (n = 36 mCherry cells, 39 β4-mCherry cells, 36 β23-mCherry cells; one-way ANOVA with Tukey's post hoc test). Scale bar in (A), 10 μm. Data information: Data in (C) are presented as box plots with whiskers indicating minimal and maximal values. Data in (D) are presented as mean ± SD. **P < 0.01; ***P < 0.001; n.s., not significant.
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The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons...
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... protein aggregates were often found in direct contact with cellular membranes, especially those of the ER (Figs 2, S3, and S4F). In some cases, ER tubes surrounded the aggregate periphery and tunnelled through its interior (Fig S4F), similar to previous observations for polyQ, α-Synuclein and heat-shock induced aggregates ( Bauerlein et al, 2017;Wagner et al, 2017;Gruber et al, 2018;Trinkaus et al, 2021). However, in contrast to polyQ fibrils ( Bauerlein et al, 2017), β protein fibrils did not appear to deform cellular membranes (Figs 2C and S3A). ...
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... similar structures were reported in Alzheimer's disease (Nixon et al, 2005; Gowrishankar et al, 2015), in Parkinson's disease, and other synucleinopathies ( Crews et al, 2010;Dehay et al, 2012;Usenovic et al, 2012) and in conditions of impaired lysosomal degradation ( Abraham et al, 1968;Spaet et al, 1983;Fernandez-Mosquera et al, 2019). Consistently with cryo-ET, light microscopy experiments with LysoTracker-loaded neurons showed that β protein expression led to an increase in lysosomal size (Fig 4A and B). Compared with mCherry cells, lysosomes with a diameter larger than 1 μm were ~3.5-fold more abundant in both β4-mCherry and β23-mCherry cells. ...
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... with mCherry cells, lysosomes with a diameter larger than 1 μm were ~3.5-fold more abundant in both β4-mCherry and β23-mCherry cells. However, the total number of LysoTracker-positive puncta per cell was reduced (Fig 4C). In agreement with cryo-ET findings, no significant colocalization was observed between β protein aggregates and LysoTracker-positive organelles (Fig 4A and D). ...
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... the total number of LysoTracker-positive puncta per cell was reduced (Fig 4C). In agreement with cryo-ET findings, no significant colocalization was observed between β protein aggregates and LysoTracker-positive organelles (Fig 4A and D). In summary, our cryo-ET and light microscopy data show that β protein aggregation leads to the accumulation of enlarged, cargorich autolysosomes, although the aggregates themselves do not appear to build up inside these organelles. ...
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... markers were not significantly different in β23-mCherry and mCherry control cells (Fig 5H-I), suggesting that autophagosome formation was not affected by β proteins. Consistently, early autophagosomes were rarely observed by cryo-ET in either control or β protein-expressing neurons (Fig S4E, numbers of observations are provided in Table S2). In contrast, real-time PCR revealed that β protein expression induced an up-regulation of transcripts of numerous lysosomal proteins (Fig 5J), a characteristic signature of aberrant lysosomal storage ( Sardiello et al, 2009;Settembre et al, 2011). ...
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... agreement with previous reports (Kantheti et al, 1998), the levels of AP-3μ1 were severely reduced in mocha cells (Fig S8A-C), consistent with the instability of the whole complex in the absence of one subunit. When mocha cells were loaded with LysoTracker Red, we observed reduced numbers of lysosomes per cell (Fig 8E and F), in line with our findings in LysoTracker-labeled β protein neurons (Fig 4C). These results confirm that impairment of the AP-3 complex leads to lysosomal defects. ...
Citations
... Without SAS, FITC and Alexa405 were co-localized in swollen vesicles ( Figure 3C, rows 2-3; Supplementary Figures S1A,B) implying that the treated FITC-Aβ 42 remained intact in cells. The morphological changes of vesicles are also consistent with previous reports that Aβ 42 induces endosomal and lysosomal swelling in neuronal cells (Riera-Tur et al., 2022). On the contrary, upon the treatment with SAS modRNA, the co-localization of FITC and Alexa405 was significantly reduced with most of the two fluorescences being separated in small-sized puncta ( Figure 3C, rows 4-5; Supplementary Figures S1A,B). ...
Excessive accumulation of amyloid-β (Aβ) has been associated with the pathogenesis of Alzheimer’s disease (AD). Clinical studies have further proven that elimination of Aβ can be a viable therapeutic option. In the current study, we conceptualized a fusion membrane protein, referred to as synthetic α-secretase (SAS), that can cleave amyloid precursor protein (APP) and Aβ specifically at the α-site. In mammalian cells, SAS indeed cleaved APP and Aβ at the α-site. Overexpression of SAS in the hippocampus was achieved by direct injection of recombinant adeno-associated virus serotype 9 (AAV9) that expresses SAS (AAV9-SAS) into the bilateral ventricles of mouse brains. SAS enhanced the non-amyloidogenic processing of APP, thus reducing the levels of soluble Aβ and plaques in the 5xFAD mice. In addition, SAS significantly attenuated the cognitive deficits in 5xFAD mice, as demonstrated by novel object recognition and Morris water maze tests. Unlike other Aβ-cleaving proteases, SAS has highly strict substrate specificity. We propose that SAS can be an efficient modality to eliminate excessive Aβ from diseased brains.
... Oligomeric species derived from αS, Aβ and Htt-53Q sharing significant structural similarities bind with high affinity and inhibit the functionality of the 20S proteasome through allosteric impairment 255 . Cryo electron tomography also revealed that amyloid-like fibril aggregates can cause gain-of-function toxicity resulting in lysosomal defects 256 and impair vesicular trafficking as shown in cases of Alzheimer disease and Parkinson disease pathology 257 . ...
Despite advances in machine learning-based protein structure prediction, we are still far from fully understanding how proteins fold into their native conformation. The conventional notion that polypeptides fold spontaneously to their biologically active states has gradually been replaced by our understanding that cellular protein folding often requires context-dependent guidance from molecular chaperones in order to avoid misfolding. Misfolded proteins can aggregate into larger structures, such as amyloid fibrils, which perpetuate the misfolding process, creating a self-reinforcing cascade. A surge in amyloid fibril structures has deepened our comprehension of how a single polypeptide sequence can exhibit multiple amyloid conformations, known as polymorphism. The assembly of these polymorphs is not a random process but is influenced by the specific conditions and tissues in which they originate. This observation suggests that, similar to the folding of native proteins, the kinetics of pathological amyloid assembly are modulated by interactions specific to cells and tissues. Here, we review the current understanding of how intrinsic protein conformational propensities are modulated by physiological and pathological interactions in the cell to shape protein misfolding and aggregation pathology.
... It appears that, as the b-sheet structure continues to expand and elongate, it absorbs energy from the surrounding fluid milieu. This would explain scientists' claim that Ab aggregates exhibit a 'toxic gain of function' [261][262][263][264]. But the so-called toxicity lies not in the inert Ab plaques themselves but in the fact that their transition to an insoluble state diverts energy away from other cell functions. ...
In this paper we examine 20th century conceptual developments regarding dementia and the NDs, from the first recognition of their clinical and pathological characteristics, through recognition of their molecular and cellular attributes and, ultimately, to recognition of their dynamic and vascular origins. We introduce a new energy based causal model of these disabling neurologic conditions that provides vital insights into their origins and necessary treatment. The term 'causal' implies that future treatment of these conditions necessarily entails recognition and correction of underlying energy deficits.
... We also observed cases of mitochondrial clustering in single neurite segments ( Figure 5D and E), something that would not be resolvable by conventional light microscopy. Lastly, multivesicular bodies 19 were seen ( Figure 9F, Figure 5D and E), which demonstrated that chemical fixation could be used to arrest cells at certain time points, which would be useful for neurodegenerative studies where the autophagy-lysosomal pathway is disrupted (Riera-Tur et al., 2022). ...
Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3 rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.
... In fact, with age and under stress, lysosomes accumulate lipofuscin, which cannot be degraded, leading to lysosomal dysfunction (Brunk and Terman, 2002;Trigo et al., 2022). Strikingly, in various lysosomal storage diseases, lysosomal defects initially produce a burden of amyloidogenic proteins (Monaco and Fraldi, 2020;Riera-Tur et al., 2022). In AD, allele ε4 of the APOE gene has been related to alterations in the endocytic, autophagic, and lysosomal processes (Schmukler et al., 2018;Eran and Ronit, 2022;Fernández-Calle et al., 2022). ...
Introduction: Alzheimer’s disease remains the most common neurodegenerative disorder, depicted mainly by memory loss and the presence in the brain of senile plaques and neurofibrillary tangles. This disease is related to several cellular alterations like the loss of synapses, neuronal death, disruption of lipid homeostasis, mitochondrial fragmentation, or raised oxidative stress. Notably, changes in the autophagic pathway have turned out to be a key factor in the early development of the disease. The aim of this research is to determine the impact of the APOE allele ε4 and G206D-PSEN1 on the underlying mechanisms of Alzheimer’s disease.
Methods: Fibroblasts from Alzheimer’s patients with APOE 3/4 + G206D-PSEN1 mutation and homozygous APOE ε4 were used to study the effects of APOE polymorphism and PSEN1 mutation on the autophagy pathway, mitochondrial network fragmentation, superoxide anion levels, lysosome clustering, and p62/SQSTM1 levels.
Results: We observed that the APOE allele ε4 in homozygosis induces mitochondrial network fragmentation that correlates with an increased colocalization with p62/SQSTM1, probably due to an inefficient autophagy. Moreover, G206D-PSEN1 mutation causes an impairment of the integrity of mitochondrial networks, triggering high superoxide anion levels and thus making APOE 3/4 + PSEN1 fibroblasts more vulnerable to cell death induced by oxidative stress. Of note, PSEN1 mutation induces accumulation and clustering of lysosomes that, along with an increase of global p62/SQSTM1, could compromise lysosomal function and, ultimately, its degradation.
Conclusion: The findings suggest that all these modifications could eventually contribute to the neuronal degeneration that underlies the pathogenesis of Alzheimer’s disease. Further research in this area may help to develop targeted therapies for the treatment of Alzheimer’s disease.
... Cryo-tilt series were acquired at ROIs 1-3, reconstructed and overlayed with the cryo-fLM z-stacks. Endolysosomes were readily discernible at each mCherry-labelled vesicle ( Fig 7F-H'; Movie S10-12) and these morphologies were consistent with other recent studies looking at lysosomal structure by cryo-FIB and cryo-ET 28,29 . To further demonstrate the utility of this method, we redistributed Rab11a-positive recycling endosomes from perinuclear regions to the cell periphery by coexpression of FuRed-Rab11a-CIBN, Kif5a-GFP-CIBN and Cry2-cluster-mCerulean. ...
Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.
... Defective autophagosome maturation has been detected in both the CLN3 mouse model and in fibroblasts derived from patients as well as in neuronal cells derived from patient-specific induced pluripotent stem cells [164,168,169,211]. Interestingly, a lack of autophagy completion in LSDs leads to the persistence of ubiquitinated and aggregate-prone polypeptides in the cytoplasm, including p62/SQSTM1, α-synuclein and Huntingtin protein [156,184,212,213]. Moreover, α-synuclein itself contributes to neurodegeneration by reducing the efficiency of autophagosome formation [214] and is also the main component of Lewy bodies that are usually elevated in Parkinson's disease and other forms of dementia. ...
Lysosomal storage diseases (LSDs) comprise a group of inherited monogenic disorders characterized by lysosomal dysfunctions due to undegraded substrate accumulation. They are caused by a deficiency in specific lysosomal hydrolases involved in cellular catabolism, or non-enzymatic proteins essential for normal lysosomal functions. In LSDs, the lack of degradation of the accumulated substrate and its lysosomal storage impairs lysosome functions resulting in the perturbation of cellular homeostasis and, in turn, the damage of multiple organ systems. A substantial number of studies on the pathogenesis of LSDs has highlighted how the accumulation of lysosomal substrates is only the first event of a cascade of processes including the accumulation of secondary metabolites and the impairment of cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis, that significantly contribute to the onset and progression of these diseases. Emerging studies on lysosomal biology have described the fundamental roles of these organelles in a variety of physiological functions and pathological conditions beyond their canonical activity in cellular waste clearance. Here, we discuss recent advances in the knowledge of cellular and molecular mechanisms linking lysosomal positioning and trafficking to LSDs.
... In the context of Alzheimer's disease, its effects include causing cholinergic dysfunction and increasing amyloid-β deposition (Chen & Yeong, 2020). Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-offunction toxicity (Riera-Tur, Schafer, Hornburg, Mishra, da Silva Padilha, Fernandez-Mosquera, et al., 2022). Four-dimensional labelfree quantification (4D LFQ) proteomics and phosphoproteomics were conducted to broaden our understanding of the cellular processes and networks involved and to explore the possible mechanisms of WNP-10. ...
This work aimed to explore the underlying mechanisms of memory improvement effects of a walnut derived peptide WNP-10. The morris water maze test, combined with ultrastructural observation, hematoxylin and eosin and Nissl staining showed that WNP–10 significantly improved the learning and memory capability of the scopolamine–injured mice. The four–dimensional label-free quantification proteomics analysis identified 88 differentially expressed proteins in the WNP–10-treated group compared with scopolamine-induced impairment group. Pathway enrichment analysis and western blotting demonstrated that the WNP-10 can regulate the phosphatidylinositol-3-phosphate 5-kinase, cathepsin L, N-acetylgalactosamine 6-sulfate sulfatase and AP-3 complex subunit mu-1 expression to affect inositol phosphate metabolism, thereby maintaining lysosome homeostasis in scopolamine–injured mice. Notably, the results of phosphoproteomics demonstrated that WNP–10 administration resulted in the increased phosphorylation of phosphatidylinositol-3-phosphate 5-kinase. These findings provide novel insights into the underlying mechanism of memory improvement of walnut peptides.
Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.
