bims-proarb Biomed News
on Proteostasis in aging and regenerative biology
Issue of 2022–01–09
thirteen papers selected by
Rich Giadone, Harvard University
  1. Assays to Study IRE1 Activation and Signaling.
  2. HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR).
  3. Indirect Methods To Measure Unfolded Proteins In Living Cells Using Fluorescent Proteins.
  4. Modulating autophagic flux via ROS-responsive targeted micelles to restore neuronal proteostasis in Alzheimer's disease.
  5. Multidimensional dynamics of the proteome in the neurodegenerative and aging mammalian brain.
  6. Effect of soluble amyloid precursor protein-alpha on adult hippocampal neurogenesis in a mouse model of Alzheimer's disease.
  7. Editorial: The Biochemistry of Amyloids in Neurodegenerative Diseases, Volume I.
  8. Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans.
  9. Generation of an induced pluripotent stem cell line (ICNDXHi001-A) from a patient with frontotemporal dementia carrying a heterozygous mutation c.796C > G (p.L266V) in MAPT.
  10. The Effect of Alzheimer's Disease-Associated Genetic Variants on Longevity.
  11. Chaperonin Mechanisms: Multiple and (Mis)Understood?
  12. Presenilin is essential for ApoE secretion, a novel role of presenilin involved in Alzheimer's disease pathogenesis.
  13. Forward steps in organoid-based forward screening.
  1. Methods Mol Biol. 2022 ;2378 141-168
    Assays to Study IRE1 Activation and Signaling.
    Paloma Moraga, Raul Aravena, Hery Urra, Claudio Hetz.
      The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.  In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.
    Keywords:  ER stress; IRE1 activation; IRE1 oligomerization; IRE1 phosphorylation; Regulated IRE1-dependent decay (RIDD); Unfolded protein response; XBP1 mRNA splicing
    DOI:  https://doi.org/10.1007/978-1-0716-1732-8_10
  2. Methods Mol Biol. 2022 ;2378 317-327
    HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR).
    Mehrnoosh Ghafouri, Chester B Gauss, Andrew M Fribley.
      The identification of small molecules and natural product extracts that enhance or interfere with the productivity of protein folding in the endoplasmic reticulum (ER) has the potential to improve a wide variety of human pathologies. Every protein that is destined for a lysosome, integral to the cell membrane, or secreted, is folded, post-translationally modified, and exported to the cytoplasm from the ER-Golgi complex. The following protocols have successfully employed several high-fidelity cell-based luciferase high-throughput screens (HTS) to identify activators and inhibitors of ER stress and the unfolded protein response (UPR).
    Keywords:  ATF4; CHOP; Cell-based assay; ER stress; GRP78/BiP; UPR; XBP1
    DOI:  https://doi.org/10.1007/978-1-0716-1732-8_20
  3. Methods Mol Biol. 2022 ;2378 31-44
    Indirect Methods To Measure Unfolded Proteins In Living Cells Using Fluorescent Proteins.
    Elisabet Navarro-Tapia, Roberto Pérez-Torrado.
      In the study of the unfolded protein response pathway, it is essential to determine the amount of unfolded proteins that the cell is accumulating. Besides being essential it is one of the most challenging technique because of the difficulty to detect unfolded proteins without producing protein denaturation with the method itself. Thus, indirect methods became very useful as the use of fluorescent proteins. In this chapter, we present some of the most used methods to indirectly measure protein folding in living cells using fluorescent proteins.
    Keywords:  Endoplasmic reticulum stress; Fluorescent proteins; Indirect method; Unfolded protein response
    DOI:  https://doi.org/10.1007/978-1-0716-1732-8_3
  4. Bioact Mater. 2022 May;11 300-316
    Modulating autophagic flux via ROS-responsive targeted micelles to restore neuronal proteostasis in Alzheimer's disease.
    Shuting Xu, Peng Yang, Kang Qian, Yixian Li, Qian Guo, Pengzhen Wang, Ran Meng, Jing Wu, Jinxu Cao, Yunlong Cheng, Minjun Xu, Qizhi Zhang.
      Compromised autophagy and defective lysosomal clearance significantly contribute to impaired neuronal proteostasis, which represents a hallmark of Alzheimer's disease (AD) and other age-related neurodegenerative disorders. Growing evidence has implicated that modulating autophagic flux, instead of inducing autophagosome formation alone, would be more reliable to rescue neuronal proteostasis. Concurrently, selectively enhancing drug concentrations in the leision areas, instead of the whole brain, will maximize therapeutic efficacy while reduing non-selective autophagy induction. Herein, we design a ROS-responsive targeted micelle system (TT-NM/Rapa) to enhance the delivery efficiency of rapamycin to neurons in AD lesions guided by the fusion peptide TPL, and facilitate its intracellular release via ROS-mediated disassembly of micelles, thereby maximizing autophagic flux modulating efficacy of rapamycin in neurons. Consequently, it promotes the efficient clearance of intracellular neurotoxic proteins, β-amyloid and hyperphosphorylated tau proteins, and ameliorates memory defects and neuronal damage in 3 × Tg-AD transgenic mice. Our studies demonstrate a promising strategy to restore autophagic flux and improve neuronal proteostasis by rationally-engineered nano-systems for delaying the progression of AD.
    Keywords:  Alzheimer's disease; Autophagic flux; Brain-neuron targeting; ROS-Responsive micelle system; Rapamycin
    DOI:  https://doi.org/10.1016/j.bioactmat.2021.09.017
  5. Mol Cell Proteomics. 2021 Dec 31. pii: S1535-9476(21)00164-X. [Epub ahead of print] 100192
    Multidimensional dynamics of the proteome in the neurodegenerative and aging mammalian brain.
    Byron Andrews, Alan E Murphy, Michele Stofella, Sarah Maslen, Leonardo Almeida-Souza, J Mark Skehel, Nathan G Skene, Frank Sobott, René A W Frank.
      The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labelling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimer's disease increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, aging in wildtype mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. Overall, this simple multidimensional approach enables the comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.
    Keywords:  Alzheimer's disease; Protein turnover; Proteomics; SILAM; neurodegeneration
    DOI:  https://doi.org/10.1016/j.mcpro.2021.100192
  6. Mol Brain. 2022 Jan 03. 15(1): 5
    Effect of soluble amyloid precursor protein-alpha on adult hippocampal neurogenesis in a mouse model of Alzheimer's disease.
    Shane M Ohline, Connie Chan, Lucia Schoderboeck, Hollie E Wicky, Warren P Tate, Stephanie M Hughes, Wickliffe C Abraham.
      Soluble amyloid precursor protein-alpha (sAPPα) is a regulator of neuronal and memory mechanisms, while also having neurogenic and neuroprotective effects in the brain. As adult hippocampal neurogenesis is impaired in Alzheimer's disease, we tested the hypothesis that sAPPα delivery would rescue adult hippocampal neurogenesis in an APP/PS1 mouse model of Alzheimer's disease. An adeno-associated virus-9 (AAV9) encoding murine sAPPα was injected into the hippocampus of 8-month-old wild-type and APP/PS1 mice, and later two different thymidine analogues (XdU) were systemically injected to label adult-born cells at different time points after viral transduction. The proliferation of adult-born cells, cell survival after eight weeks, and cell differentiation into either neurons or astrocytes was studied. Proliferation was impaired in APP/PS1 mice but was restored to wild-type levels by viral expression of sAPPα. In contrast, sAPPα overexpression failed to rescue the survival of XdU+-labelled cells that was impaired in APP/PS1 mice, although it did cause a significant increase in the area density of astrocytes in the granule cell layer across both genotypes. Finally, viral expression of sAPPα reduced amyloid-beta plaque load in APP/PS1 mice in the dentate gyrus and somatosensory cortex. These data add further evidence that increased levels of sAPPα could be therapeutic for the cognitive decline in AD, in part through restoration of the proliferation of neural progenitor cells in adults.
    Keywords:  APP/PS1; Adult neurogenesis; Alzheimer’s disease; Astrocytes; Dentate gyrus; Differentiation; Proliferation; sAPPα
    DOI:  https://doi.org/10.1186/s13041-021-00889-1
  7. Front Neurosci. 2021 ;15 819481
    Editorial: The Biochemistry of Amyloids in Neurodegenerative Diseases, Volume I.
    Cláudio M Gomes, Wolfgang Hoyer, Jinghui Luo.
      
    Keywords:  Alzheimer's disease; Parkinson's disease; SOD1; aggregation inhibitors; amyloid beta; protein aggregation; synuclein; tau
    DOI:  https://doi.org/10.3389/fnins.2021.819481
  8. J Vis Exp. 2021 Dec 17.
    Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans.
    Jelle Molenkamp, Anna den Outer, Vera van Schijndel, Tessa Sinnige.
      Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process. However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion. Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.
    DOI:  https://doi.org/10.3791/63365
  9. Stem Cell Res. 2022 Jan 03. pii: S1873-5061(22)00003-4. [Epub ahead of print]59 102654
    Generation of an induced pluripotent stem cell line (ICNDXHi001-A) from a patient with frontotemporal dementia carrying a heterozygous mutation c.796C > G (p.L266V) in MAPT.
    Huimin Cai, Yana Pang, Longfei Jia.
      Frontotemporal dementia (FTD) caused by microtubule-associated protein tau (MAPT) mutations is not rare and is almost fully penetrant. However, no disease-modifying treatment for FTD is currently available. Here, we demonstrated the establishment and characterization of a novel human induced pluripotent stem cell (iPSC) line ICNDXHi001-A from a patient with FTD carrying genetic variant MAPT c.796C > G (p.L266V). The generated cell line showed trilineage differentiation potential, expression of pluripotency markers, a normal karyotype, and retention of MAPT mutation. The study provides a useful model to further elucidate the underlying mechanisms of FTD and to facilitate novel therapy development.
    DOI:  https://doi.org/10.1016/j.scr.2022.102654
  10. Front Genet. 2021 ;12 748781
    The Effect of Alzheimer's Disease-Associated Genetic Variants on Longevity.
    Niccolò Tesi, Marc Hulsman, Sven J van der Lee, Iris E Jansen, Najada Stringa, Natasja M van Schoor, Philip Scheltens, Wiesje M van der Flier, Martijn Huisman, Marcel J T Reinders, Henne Holstege.
      Human longevity is influenced by the genetic risk of age-related diseases. As Alzheimer's disease (AD) represents a common condition at old age, an interplay between genetic factors affecting AD and longevity is expected. We explored this interplay by studying the prevalence of AD-associated single-nucleotide-polymorphisms (SNPs) in cognitively healthy centenarians, and replicated findings in a parental-longevity GWAS. We found that 28/38 SNPs that increased AD-risk also associated with lower odds of longevity. For each SNP, we express the imbalance between AD- and longevity-risk as an effect-size distribution. Based on these distributions, we grouped the SNPs in three groups: 17 SNPs increased AD-risk more than they decreased longevity-risk, and were enriched for β-amyloid metabolism and immune signaling; 11 variants reported a larger longevity-effect compared to their AD-effect, were enriched for endocytosis/immune-signaling, and were previously associated with other age-related diseases. Unexpectedly, 10 variants associated with an increased risk of AD and higher odds of longevity. Altogether, we show that different AD-associated SNPs have different effects on longevity, including SNPs that may confer general neuro-protective functions against AD and other age-related diseases.
    Keywords:  aging; alzheimer’s disease; centenarians; cognitively healthy; effect on aging; protective variants
    DOI:  https://doi.org/10.3389/fgene.2021.748781
  11. Annu Rev Biophys. 2022 Jan 04.
    Chaperonin Mechanisms: Multiple and (Mis)Understood?
    Amnon Horovitz, Tali Haviv Reingewertz, Jorge Cuéllar, José María Valpuesta.
      The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biophys-082521-113418
  12. J Neurosci. 2022 Jan 05. pii: JN-RM-2039-21. [Epub ahead of print]
    Presenilin is essential for ApoE secretion, a novel role of presenilin involved in Alzheimer's disease pathogenesis.
    Sadequl Islam, Yang Sun, Yuan Gao, Tomohisa Nakamura, Arshad Ali Noorani, Tong Li, Philip C Wong, Noriyuki Kimura, Etsuro Matsubara, Kensaku Kasuga, Takeshi Ikeuchi, Taisuke Tomita, Kun Zou, Makoto Michikawa.
      Alzheimer disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-β-protein (Aβ) into amyloid plaques in patient brain. Mutations in presenilin (PS) lead to abnormal generation of Aβ, which is the major cause of familial AD (FAD) and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin (NCT) deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear-transport receptor. Moreover, expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal controls. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENTFAD typically results from mutations in the genes encoding amyloid precursor protein (APP), PS1 or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased Aβ42/Aβ40 ratio, which induce early amyloid deposition and FAD. On the other hand, ApoE4 is the major genetic risk factor for SAD and contributes to AD pathogenesis because it has reduced Aβ clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms, however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.
    DOI:  https://doi.org/10.1523/JNEUROSCI.2039-21.2021
  13. Cell Stem Cell. 2022 Jan 06. pii: S1934-5909(21)00488-4. [Epub ahead of print]29(1): 7-8
    Forward steps in organoid-based forward screening.
    Melissa H Little, Sara E Howden.
      In this issue of Cell Stem Cell, Ungricht et al. (2022) perform a temporally controlled CRISPR/Cas9-based genome-wide screen in kidney organoids to uncover key gene networks important for the specification of kidney cell types from human pluripotent stem cells, thus furthering our understanding of human kidney development and disease.
    DOI:  https://doi.org/10.1016/j.stem.2021.12.004
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