bims-proarb 2021-06-27 papers

bims-proarb

Biomed News

on Proteostasis in aging and regenerative biology

Issue of 2021–06–27
twenty-two papers selected by
Rich Giadone, Harvard University

The Cross-Links of Endoplasmic Reticulum Stress, Autophagy, and Neurodegeneration in Parkinson's Disease.
Hepatic X-box binding protein 1 and unfolded protein response is impaired in weanling mice with resultant hepatic injury.
Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans.
Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons.
ER stress associated TXNIP-NLRP3 inflammasome activation in hippocampus of human Alzheimer's disease.
The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.
Aging and Mesenchymal Stem Cells: Therapeutic Opportunities and Challenges in the Older Group.
Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.
HSP70-16 and VDAC3 jointly inhibit seed germination under cold stress in Arabidopsis.
The Hsp70-Chaperone Machines in Bacteria.
Global Single Clustering of Phenotype-Associated Human Aging Genes in the Co-Expression and Physical Interaction Networks: An OMIM-Based Investigative Review.
Aggregation and partial immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells.
Dysregulation of the secretory pathway connects Alzheimer's disease genetics to aggregate formation.
Soluble Amyloid-β Consumption in Alzheimer's Disease.
Transmembrane dislocases: a second chance for protein targeting.
Proteasomal conformation controls unfolding ability.
The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery.
Diterpene glycosides from Holothuria scabra exert the α-synuclein degradation and neuroprotection against α-synuclein-Mediated neurodegeneration in C. elegans model.
Size-based Degradation of Therapeutic Proteins - Mechanisms, Modelling and Control.
ATF4-dependent heme-oxygenase-1 attenuates diabetic nephropathy by inducing autophagy and inhibiting apoptosis in podocyte.
High intensity interval training induces dysregulation of mitochondrial respiratory complex and mitophagy in the hippocampus of middle-aged mice.
Generation and validation of APOE knockout human iPSC-derived cerebral organoids.

Front Aging Neurosci. 2021 ;13 691881

The Cross-Links of Endoplasmic Reticulum Stress, Autophagy, and Neurodegeneration in Parkinson's Disease.

Haigang Ren, Wanqing Zhai, Xiaojun Lu, Guanghui Wang.

  Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and it is characterized by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), as well as the presence of intracellular inclusions with α-synuclein as the main component in surviving DA neurons. Emerging evidence suggests that the imbalance of proteostasis is a key pathogenic factor for PD. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and autophagy, two major pathways for maintaining proteostasis, play important roles in PD pathology and are considered as attractive therapeutic targets for PD treatment. However, although ER stress/UPR and autophagy appear to be independent cellular processes, they are closely related to each other. In this review, we focused on the roles and molecular cross-links between ER stress/UPR and autophagy in PD pathology. We systematically reviewed and summarized the most recent advances in regulation of ER stress/UPR and autophagy, and their cross-linking mechanisms. We also reviewed and discussed the mechanisms of the coexisting ER stress/UPR activation and dysregulated autophagy in the lesion regions of PD patients, and the underlying roles and molecular crosslinks between ER stress/UPR activation and the dysregulated autophagy in DA neurodegeneration induced by PD-associated genetic factors and PD-related neurotoxins. Finally, we indicate that the combined regulation of ER stress/UPR and autophagy would be a more effective treatment for PD rather than regulating one of these conditions alone.

Keywords:  ER stress; Parkinson’s disease; UPR; autophagy; cross-link; α-synuclein

DOI:  https://doi.org/10.3389/fnagi.2021.691881
Hepatology. 2021 Jun 25.

Hepatic X-box binding protein 1 and unfolded protein response is impaired in weanling mice with resultant hepatic injury.

Alyssa Kriegermeier, Angela Hyon, Meredith Sommars, Susan Hubchak, Brian LeCuyer, Xiaoying Liu, Grant Barish, Richard M Green.

   BACKGROUND & AIMS: The unfolded protein response (UPR) is a coordinated cellular response to endoplasmic reticulum (ER) stress that functions to maintain cellular homeostasis. When ER stress is unresolved, the UPR can trigger apoptosis. Pathways within the UPR influence bile acid metabolism in adult animal models and adult human liver diseases, however the UPR has not been studied in young animal models or pediatric liver diseases. In this study we sought to determine if weanling age mice had altered UPR activation compared to adult mice which could lead to increased bile acid induced hepatic injury APPROACH & RESULTS: We demonstrate that after 7 days of cholic acid (CA) feeding to wild type (WT) animals, weanling age mice have a 2-fold greater serum ALT levels compared to adult mice, with increased hepatic apoptosis. Weanling mice fed CA have increased hepatic nuclear X-box binding protein 1 spliced (XBP1s) expression, but cannot increase expression of its protective downstream targets endoplasmic reticulum DNA J domain-containing protein 4 (ERdj4) and ER degradation enhancing α-mannoside (EDEM). In response to tunicamycin induced ER stress, young mice have blunted expression of several UPR pathways compared to adult mice. CA feeding to adult liver-specific XBP1 knockout (LS-XBP1-/- ) mice, which are unable to resolve hepatic ER stress, leads to increased serum ALT and C/EBP homologous protein (CHOP), a proapoptotic UPR molecule, expression to levels similar to CA fed LS-XBP1-/- weanlings.
CONCLUSIONS: Weanling mice have attenuated hepatic XBP1 signaling and impaired UPR activation with resultant increased susceptibility to bile acid induced injury.

Keywords:  ER stress; bile acid; cholestasis; endoplasmic reticulum; liver

DOI:  https://doi.org/10.1002/hep.32031
Sci Adv. 2021 Jun;pii: eabg3012. [Epub ahead of print]7(26):

Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans.

Giuseppe Calculli, Hyun Ju Lee, Koning Shen, Uyen Pham, Marija Herholz, Aleksandra Trifunovic, Andrew Dillin, David Vilchez.

Protein aggregation causes intracellular changes in neurons, which elicit signals to modulate proteostasis in the periphery. Beyond the nervous system, a fundamental question is whether other organs also communicate their proteostasis status to distal tissues. Here, we examine whether proteostasis of the germ line influences somatic tissues. To this end, we induce aggregation of germline-specific PGL-1 protein in germline stem cells of Caenorhabditis elegans Besides altering the intracellular mitochondrial network of germline cells, PGL-1 aggregation also reduces the mitochondrial content of somatic tissues through long-range Wnt signaling pathway. This process induces the unfolded protein response of the mitochondria in the soma, promoting somatic mitochondrial fragmentation and aggregation of proteins linked with neurodegenerative diseases such as Huntington's and amyotrophic lateral sclerosis. Thus, the proteostasis status of germline stem cells coordinates mitochondrial networks and protein aggregation through the organism.

DOI: https://doi.org/10.1126/sciadv.abg3012
STAR Protoc. 2021 Jun 18. 2(2): 100580

Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons.

Aiqun Li, Samuel Cartwright, Alex Yu, Seok-Man Ho, Nadine Schrode, P J Michael Deans, Marliette R Matos, Meilin Fernandez Garcia, Kayla G Townsley, Bin Zhang, Kristen J Brennand.

  We describe a CRISPR inhibition (CRISPRi) protocol to repress endogenous gene expression (e.g., ATP6V1A) in human induced pluripotent stem cell-derived NGN2-induced glutamatergic neurons. CRISPRi enables efficient and precise gene repression of one or multiple target genes via delivering gRNA(s) to direct a dCas9-KRAB fusion protein to the gene(s) of interest. This protocol can also be adapted for gene activation and high-throughput gene manipulation, allowing assessment of the transcriptomic and phenotypic impact of candidate gene(s) associated with neurodevelopment or brain disease. For complete details on the use and execution of this protocol, please refer to Ho et al. (2017) and Wang et al. (2021).

Keywords:  CRISPR; Cell Differentiation; Molecular Biology; Neuroscience; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2021.100580
Neurochem Int. 2021 Jun 18. pii: S0197-0186(21)00150-9. [Epub ahead of print] 105104

ER stress associated TXNIP-NLRP3 inflammasome activation in hippocampus of human Alzheimer's disease.

Saifudeen Ismael, Wajidunnisa, Kazuko Sakata, Michael P McDonald, Francesca-Fang Liao, Tauheed Ishrat.

  Although the exact etiology of Alzheimer's disease (AD) is poorly understood, experimental and clinical evidences suggest the contribution of neuroinflammation in the pathogenesis of AD. Pathologically, AD brain is characterized by imbalance in redox status, elevated endoplasmic reticulum (ER) stress, synaptic dysfunction, inflammation and progressive neurodegeneration. It has been noted that continuous accumulation of amyloid-beta (Aβ) and intracellular neurofibrillary tangles (NFTs) in AD brain trigger ER stress, which contributes to neurodegeneration. Similarly, experimental evidences support the hypothesis that thioredoxin-interacting protein (TXNIP), an endogenous regulator of redox regulator thioredoxin (TRX) is activated by ER stress and contributes to activation of NLRP3 (NOD-like receptor protein 3) inflammatory cascade in hippocampus of the AD brain. Hippocampus of postmortem human AD and aged matched non-AD controls were analyzed for the expression ER stress markers and TXNIP-NLRP3 inflammasome at cellular and molecular levels. We found higher expression of TXNIP at protein and transcript level in close association with pathological markers of AD such as Aβ and NFTs in AD hippocampus. In addition, our results demonstrated that TXNIP was co-localized in neurons and microglia. Moreover, expression of binding immunoglobulin protein (BIP), activated eukaryotic initiation factor-2α (eIf2α) and C/EBP homology protein (CHOP), proteins involved the development of ER stress were elevated in AD hippocampus. Further elevated expression of effector molecules of NLRP3 inflammasome activation such as apoptosis associated speck-like protein (ASC), cleaved caspase-1 and cleaved interleukin-1β were observed in the AD hippocampus. The study suggests that TXNIP could be a link that connect ER stress with neuroinflammation. Thus, TXNIP can be a possible therapeutic target to mitigate the progression of neuroinflammation in the pathogenesis of AD.

Keywords:  Alzheimer’s disease; Endoplasmic reticulum stress; NLRP3 inflammasome; Thioredoxin-interacting protein

DOI:  https://doi.org/10.1016/j.neuint.2021.105104
Sci Rep. 2021 Jun 22. 11(1): 13086

The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.

Dinh Thi Nguyen, Thuong Manh Le, Tsuyoshi Hattori, Mika Takarada-Iemata, Hiroshi Ishii, Jureepon Roboon, Takashi Tamatani, Takayuki Kannon, Kazuyoshi Hosomichi, Atsushi Tajima, Shusuke Taniuchi, Masato Miyake, Seiichi Oyadomari, Takashi Tanaka, Nobuo Kato, Shunsuke Saito, Kazutoshi Mori, Osamu Hori.

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

DOI: https://doi.org/10.1038/s41598-021-92529-w
Gerontology. 2021 Jun 23. 1-14

Aging and Mesenchymal Stem Cells: Therapeutic Opportunities and Challenges in the Older Group.

Huan Chen, Ousheng Liu, Sijia Chen, Yueying Zhou.

  With aging, a portion of cells, including mesenchymal stem cells (MSCs), become senescent, and these senescent cells accumulate and promote various age-related diseases. Therefore, the older age group has become a major population for MSC therapy, which is aimed at improving tissue regeneration and function of the aged body. However, the application of MSC therapy is often unsatisfying in the aged group. One reasonable conjecture for this correlation is that aging microenvironment reduces the number and function of MSCs. Cellular senescence also plays an important role in MSC function impairment. Thus, it is necessary to explore the relationship between senescence and MSCs for improving the application of MSCs in the elderly. Here, we present the influence of aging on MSCs and the characteristics and functional changes of senescent MSCs. Furthermore, current therapeutic strategies for improving MSC therapy in the elderly group are also discussed.

Keywords:  Aged group; Antiaging; Cellular senescence; Mesenchymal stem cell therapy; Mesenchymal stem cells

DOI:  https://doi.org/10.1159/000516668
Neurochem Res. 2021 Jun 24.

Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Areechun Sotthibundhu, Chutikorn Nopparat, Sitakan Natphopsuk, Sophida Phuthong, Parinya Noisa, Piyarat Govitrapong.

  Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase (JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

Keywords:  Autophagy; Direct reprogramming; Fibroblast; Melatonin

DOI:  https://doi.org/10.1007/s11064-021-03382-2
Plant Cell Environ. 2021 Jun 25.

HSP70-16 and VDAC3 jointly inhibit seed germination under cold stress in Arabidopsis.

Muhammad Ashraf, Qionglei Mao, Jun Hong, Lei Shi, Xiaoruo Ran, Fiza Liaquat, Muhammad Uzair, Wanqi Liang, Alisdair R Fernie, Jianxin Shi.

  Abscisic acid (ABA) transport plays a crucial role in seed germination under unfavorable conditions such as cold stress. Heat shock protein 70 (HSP70) and voltage-dependent anion channel (VDAC) protein are both involved in cold stress responses in Arabidopsis. However, their roles in seed germination with regard to ABA signaling remains unknown. Here we demonstrated that Arabidopsis HSP70-16 and VDAC3 jointly suppress seed germination under cold stress conditions. At 4o C, both HSP70-16 and VDAC3 facilitated the efflux of ABA from the endosperm to the embryo and thus inhibited seed germination. HSP70-16 interacted with VDAC3 on the plasma membrane and in the nucleus, and the interplay between HSP70-16 and VDAC3 activated the opening of the VDAC3 ion channel. Our work established a novel function of HSP70-16 in seed germination under cold stress and a possible association of VDAC3 activity with ABA transportation from endosperm to embryo under cold stress conditions. This article is protected by copyright. All rights reserved.

Keywords:  ABA transport; Cold stress; Heat shock protein; Seed germination; Voltage-dependent anion channel protein

DOI:  https://doi.org/10.1111/pce.14138
Front Mol Biosci. 2021 ;8 694012

The Hsp70-Chaperone Machines in Bacteria.

Matthias P Mayer.

  The ATP-dependent Hsp70s are evolutionary conserved molecular chaperones that constitute central hubs of the cellular protein quality surveillance network. None of the other main chaperone families (Tig, GroELS, HtpG, IbpA/B, ClpB) have been assigned with a comparable range of functions. Through a multitude of functions Hsp70s are involved in many cellular control circuits for maintaining protein homeostasis and have been recognized as key factors for cell survival. Three mechanistic properties of Hsp70s are the basis for their high versatility. First, Hsp70s bind to short degenerate sequence motifs within their client proteins. Second, Hsp70 chaperones switch in a nucleotide-controlled manner between a state of low affinity for client proteins and a state of high affinity for clients. Third, Hsp70s are targeted to their clients by a large number of cochaperones of the J-domain protein (JDP) family and the lifetime of the Hsp70-client complex is regulated by nucleotide exchange factors (NEF). In this review I will discuss advances in the understanding of the molecular mechanism of the Hsp70 chaperone machinery focusing mostly on the bacterial Hsp70 DnaK and will compare the two other prokaryotic Hsp70s HscA and HscC with DnaK.

Keywords:  HscA; HscC; Hsp70; allostery; molecular chaperone; protein folding; stress response

DOI:  https://doi.org/10.3389/fmolb.2021.694012
Arch Gerontol Geriatr. 2021 Jun 15. pii: S0167-4943(21)00124-2. [Epub ahead of print]96 104461

Global Single Clustering of Phenotype-Associated Human Aging Genes in the Co-Expression and Physical Interaction Networks: An OMIM-Based Investigative Review.

Rahimi M, Arabfard M, Borna H, Ohadi M.

   BACKGROUND: While a large wealth of literature on aging pertains to in silico, experimental, and predicted genes, many of those genes do not have validated phenotypic consequences in human. Online Mendelian Inheritance in Man (OMIM) provides an exceptional compendium of authoritative, validated aging genes and phenotypes, the interactions among which may enhance the overall perspective of aging mechanisms in human.
METHODS: Here, we reviewed and investigated the global clustering pattern of the OMIM-indexed aging genes (until April 2021) in the gene co-expression and physical interaction networks, using the two keywords "aging" and "ageing". To allow for validity check, we randomly selected six sets of genes from the human genome as control genes, each set consisting of a similar number of genes obtained from the OMIM search. STRING was implemented in the weighted setting and using the edge betweenness parameter, to construct the integrated and tissue-specific networks of the age-related and control genes.
RESULTS: 286 aging (ageing) genes and a wide spectrum of 96 associated phenotypes were detected, including late-onset neurodegenerative disorders, cancers, osteoarthritis, and longevity. Despite the general terms used and the vast range of age-related phenotypes, we detected single clustering of the OMIM-extracted aging (ageing) genes in each of the integrated weighted co-expression and physical interaction networks (p<0.0005), as opposed to multiple clustering of the control genes (p≥0.04). TP53 was the overlapping hub gene in each of the networks. Three genes, TP53, APP, and SIRT1 were the consistent hub genes co-expressed across eleven selected human tissues frequently affected by age-related phenotypes.
CONCLUSION: We propose predominant single clustering of the human phenotype-associated aging genes in the co-expression and physical interaction networks, and list the top pathways and genes involved.

Keywords:  OMIM; ageing; aging; clustering; human; phenotype

DOI:  https://doi.org/10.1016/j.archger.2021.104461
FEBS Lett. 2021 Jun 23.

Aggregation and partial immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

Iztok Urbančič, Lisa Schiffelers, Edward Jenkins, Weijian Gong, Ana Mafalda Santos, Falk Schneider, Caitlin O'Brien-Ball, Mai Tuyet Vuong, Nicole Ashman, Erdinc Sezgin, Christian Eggeling.

  To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein co-localisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, e.g. in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Keywords:  Diffusion; Environment-sensitive probes; Fluorescence microscopy; Membrane organisation; Spectral imaging

DOI:  https://doi.org/10.1002/1873-3468.14153
Cell Syst. 2021 Jun 18. pii: S2405-4712(21)00208-8. [Epub ahead of print]

Dysregulation of the secretory pathway connects Alzheimer's disease genetics to aggregate formation.

Chih-Chung Kuo, Austin W T Chiang, Hratch M Baghdassarian, Nathan E Lewis.

  Amyloid disorders such as Alzheimer's disease (AD) involve the aggregation of secreted proteins. However, it is largely unclear how secretory-pathway proteins contribute to amyloid formation. We developed a systems biology framework integrating expression data with protein-protein interaction networks to estimate a tissue's fitness for producing specific secreted proteins and analyzed the fitness of the secretory pathway of various brain regions and cell types for synthesizing the AD-associated amyloid precursor protein (APP). While key amyloidogenic pathway components were not differentially expressed in AD brains, we found Aβ deposition correlates with systemic down- and upregulation of the secretory-pathway components proximal to APP and amyloidogenic secretases, respectively, in AD. Our analyses suggest that perturbations from three AD risk loci cascade through the APP secretory-support network and into the endocytosis pathway, connecting amyloidogenesis to dysregulation of secretory-pathway components supporting APP and suggesting novel therapeutic targets for AD. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  late-onset Alzheimer’s; protein secretory pathway; systems biology

DOI:  https://doi.org/10.1016/j.cels.2021.06.001
J Alzheimers Dis. 2021 Jun 12.

Soluble Amyloid-β Consumption in Alzheimer's Disease.

Alberto J Espay, Andrea Sturchio, Lon S Schneider, Kariem Ezzat.

  Brain proteins function in their soluble, native conformation and cease to function when transformed into insoluble aggregates, also known as amyloids. Biophysically, the soluble-to-insoluble phase transformation represents a process of polymerization, similar to crystallization, dependent on such extrinsic factors as concentration, pH, and a nucleation surface. The resulting cross-β conformation of the insoluble amyloid is markedly stable, making it an unlikely source of toxicity. The spread of brain amyloidosis can be fully explained by mechanisms of spontaneous or catalyzed polymerization and phase transformation instead of active replication, which is an enzyme- and energy-requiring process dependent on a specific nucleic acid code for the transfer of biological information with high fidelity. Early neuronal toxicity in Alzheimer's disease may therefore be mediated to a greater extent by a reduction in the pool of soluble, normal-functioning protein than its accumulation in the polymerized state. This alternative loss-of-function hypothesis of pathogenicity can be examined by assessing the clinical and neuroimaging effects of administering non-aggregating peptide analogs to replace soluble amyloid-β levels above the threshold below which neuronal toxicity may occur. Correcting the depletion of soluble amyloid-β, however, would only exemplify 'rescue medicine.' Precision medicine will necessitate identifying the pathogenic factors catalyzing the protein aggregation in each affected individual. Only then can we stratify patients for etiology-specific treatments and launch precision medicine for Alzheimer's disease and other neurodegenerative disorders.

Keywords:  Alzheimer’s disease; clinico-pathologic; disease modification; neuroprotection; precision medicine

DOI:  https://doi.org/10.3233/JAD-210415
Trends Cell Biol. 2021 Jun 16. pii: S0962-8924(21)00099-4. [Epub ahead of print]

Transmembrane dislocases: a second chance for protein targeting.

Verena Dederer, Marius K Lemberg.

  Precise distribution of proteins is essential to sustain the viability of cells. A complex network of protein synthesis and targeting factors cooperate with protein quality control systems to ensure protein homeostasis. Defective proteins are inevitably degraded by the ubiquitin-proteasome system and lysosomes. However, due to overlapping targeting information and limited targeting fidelity, certain proteins become mislocalized. In this review, we present the idea that transmembrane dislocases recognize and remove mislocalized membrane proteins from cellular organelles. This enables other targeting attempts and prevents degradation of mislocalized but otherwise functional proteins. These transmembrane dislocases can be found in the outer mitochondrial membrane (OMM) and endoplasmic reticulum (ER). We highlight common principles regarding client recognition and outline open questions in our understanding of transmembrane dislocases.

Keywords:  AAA-ATPase Msp1/ATAD1/Thorase; ER-associated degradation; P5-type ATPase Spf1/ATP13A1; mitochondrial protein quality control; protein homeostasis; protein quality control

DOI:  https://doi.org/10.1016/j.tcb.2021.05.007
Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2101004118. [Epub ahead of print]118(25):

Proteasomal conformation controls unfolding ability.

Julianna R Cresti, Abramo J Manfredonia, Christopher E Bragança, Joseph A Boscia, Christina M Hurley, Mary D Cundiff, Daniel A Kraut.

  The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome's various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome's ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome's conformational state and its unfolding ability.

Keywords:  ATP-dependent protease; ATPases associated with diverse cellular activities (AAA); proteasome; protein degradation; protein unfolding

DOI:  https://doi.org/10.1073/pnas.2101004118
Mol Biol Cell. 2021 Jun 23. mbcE21040169

The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery.

Amrita Khakurel, Tetyana Kudlyk, Juan S Bonifacino, Vladimir V Lupashin.

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].

DOI: https://doi.org/10.1091/mbc.E21-04-0169
J Ethnopharmacol. 2021 Jun 17. pii: S0378-8741(21)00576-6. [Epub ahead of print]279 114347

Diterpene glycosides from Holothuria scabra exert the α-synuclein degradation and neuroprotection against α-synuclein-Mediated neurodegeneration in C. elegans model.

Pawanrat Chalorak, Nilubon Sornkaew, Preeyanuch Manohong, Nakorn Niamnont, Nawaphat Malaiwong, Tanapol Limboonreung, Prasert Sobhon, Michael Aschner, Krai Meemon.

   ETHNOPHARMACOLOGICAL RELEVANCE: Holothuria (Metriatyla) scabra Jaeger (H. scabra), sea cucumber, is the marine organism that has been used as traditional food and medicine to gain the health benefits since ancient time. Although our recent studies have shown that crude extracts from H. scabra exhibited neuroprotective effects against Parkinson's disease (PD), the underlying mechanisms and bioactive compounds are still unknown.
AIM OF THE STUDY: In the present study, we examined the efficacy of purified compounds from H. scabra and their underlying mechanism on α-synuclein degradation and neuroprotection against α-synuclein-mediated neurodegeneration in a transgenic Caenorhabditis elegans PD model.
MATERIAL AND METHODS: The H. scabra compounds (HSEA-P1 and P2) were purified and examined for their toxicity and optimal dose-range by food-clearance and lifespan assays. The α-synuclein degradation and neuroprotection against α-synuclein-mediated neurodegeneration were determined using transgenic C. elegans model, Punc-54::α-syn and Pdat-1:: α-syn; Pdat-1::GFP, respectively, and then further investigated by determining the behavioral assays including locomotion rate, basal slowing rate, ethanol avoidance, and area-restricted searching. The underlying mechanisms related to autophagy were clarified by quantitative PCR and RNAi experiments.
RESULTS: Our results showed that HSEA-P1 and HSEA-P2 significantly diminished α-synuclein accumulation, improved motility deficits, and recovered the shortened lifespan. Moreover, HSEA-P1 and HSEA-P2 significantly protected dopaminergic neurons from α-synuclein toxicity and alleviated dopamine-associated behavioral deficits, i.e., basal slowing, ethanol avoidance, and area-restricted searching. HSEA-P1 and HSEA-P2 also up-regulated autophagy-related genes, including beclin-1/bec-1, lc-3/lgg-1, and atg-7/atg-7. RNA interference (RNAi) of these genes in transgenic α-synuclein worms confirmed that lc-3/lgg-1 and atg-7/atg-7 were required for α-synuclein degradation and DAergic neuroprotection activities of HSEA-P1 and HSEA-P2. NMR and mass spectrometry analysis revealed that the HSEA-P1 and HSEA-P2 contained diterpene glycosides.
CONCLUSION: These findings indicate that diterpene glycosides extracted from H. scabra decreases α-synuclein accumulation and protects α-synuclein-mediated DAergic neuronal loss and its toxicities via lgg-1 and atg-7.

Keywords:  Caenorhabditis elegans; Diterpene glycoside; Dopaminergic neuron; Holothuria scabra; Parkinson's disease; α-synuclein

DOI:  https://doi.org/10.1016/j.jep.2021.114347
Biomol Concepts. 2021 Jun 19. 12(1): 68-84

Size-based Degradation of Therapeutic Proteins - Mechanisms, Modelling and Control.

Rohit Bansal, Saurabh Kumar Jha, Niraj Kumar Jha.

  Protein therapeutics are in great demand due to their effectiveness towards hard-to-treat diseases. Despite their high demand, these bio-therapeutics are very susceptible to degradation via aggregation, fragmentation, oxidation, and reduction, all of which are very likely to affect the quality and efficacy of the product. Mechanisms and modelling of these degradation (aggregation and fragmentation) pathways is critical for gaining a deeper understanding of stability of these products. This review aims to provide a summary of major developments that have occurred towards unravelling the mechanisms of size-based protein degradation (particularly aggregation and fragmentation), modelling of these size-based degradation pathways, and their control. Major caveats that remain in our understanding and control of size-based protein degradation have also been presented and discussed.

Keywords:  Protein stability; aggregation; excipients; mechanism; modelling

DOI:  https://doi.org/10.1515/bmc-2021-0008
Ren Fail. 2021 Dec;43(1): 968-979

ATF4-dependent heme-oxygenase-1 attenuates diabetic nephropathy by inducing autophagy and inhibiting apoptosis in podocyte.

Shizhu Yuan, Xudong Liang, Wenfang He, Mingzhu Liang, Juan Jin, Qiang He.

   AIM: Podocyte injury plays an important role in diabetic nephropathy (DN), yet the underlying molecular mechanisms of podocyte injury in DN is not clear. Here, we investigated the role of activating transcription factor 4 (ATF4) and HO-1 in DN-induced podocyte injury.
METHODS: Protein expression was measured by western blotting (WB) and immunofluorescence. Cellular apoptosis was quantified by flow cytometry. ATF4 siRNA knockdown and HO-1 overexpression in podocyte were employed to evaluate the role of ER stress in DN-induced apoptosis and autophagy response. Urinary protein levels, nephrin expression, serum creatinine and BUN were evaluated and glomerulosclerosis was quantified by Periodic Acid-Schiff staining.
RESULTS: Expression of ATF4 was increased in podocytes exposed to serum from DN mice. ATF4 knockdown enhanced DN-induced podocyte apoptosis. HO-1 overexpression reduced the decline of DN-induced podocyte autophagy and inhibited apoptosis and the beneficial effects of HO-1 overexpression in DN were blocked by ATF4 knockdown. The diabetic mice were significantly ameliorated by HO-1 agonist hemin treatment.
CONCLUSIONS: ATF4 induces autophagy by enhancing the expression of HO-1, and inhibits podocyte apoptosis in DN. Treatment with the HO-1 agonist reduced proteinuria, apoptosis, and enhanced autophagy response, and thus improved renal function in DN mice.

Keywords:  ATF4; HO-1; Podocyte; autophagy; diabetic nephropathy

DOI:  https://doi.org/10.1080/0886022X.2021.1936040
Behav Brain Res. 2021 Jun 17. pii: S0166-4328(21)00272-2. [Epub ahead of print] 113384

High intensity interval training induces dysregulation of mitochondrial respiratory complex and mitophagy in the hippocampus of middle-aged mice.

Yu Zhang, Bo Liao, Shuai Hu, Shan-Yao Pan, Gui-Ping Wang, Yu-Long Wang, Zheng-Hong Qin, Li Luo.

  Autophagy is involved in aging-related cognitive impairment. Aerobic exercise training can improve cognitive function in the elderly and this effect may be associated with autophagic mechanisms and mitochondrial respiratory function. High intensity interval training (HIIT) has beneficial effects on heart and skeletal muscles by activating autophagy and/or mitophagy, but the effects of HIIT on autophagy/mitophagy in the aging brain are unknown. This study investigated the effects of HIIT on the mitochondrial respiratory complex and autophagy/mitophagy, and its relation to brain function. Thirteen middle-aged male ICR mice underwent HIIT for 7 weeks. The exercise program reduced the spontaneous behavior and exploration activities of the mice. The phosphorylation level of cAMP response element binding protein (CREB) and the protein expression of brain-derived neurotrophic factor (BDNF) decreased after the 7-week HIIT. Exercise downregulated the protein expression of Complex Ⅰ and upregulated the protein expression of Complex Ⅲ, Complex Ⅳ and Complex Ⅴ. HIIT also decreased the expression of mitophagy-related proteins in the mitochondrial fractions of the hippocampus. However, HIIT did not change the expression of autophagy-related proteins LC3, P62, Atg5, Atg7, Beclin-1 and Lamp2 in the total lysate of the hippocampus. These data indicated that HIIT might have negative effects on the plasticity of the hippocampus in middle-aged mice. The effects may be related to the dysregulation of CREB-BDNF signaling, mitochondrial respiratory complex and mitophagy induced by HIIT.

Keywords:  Autophagy; High intensity interval training; Hippocampus; Mitochondrial respiratory complex; Mitophagy

DOI:  https://doi.org/10.1016/j.bbr.2021.113384
STAR Protoc. 2021 Jun 18. 2(2): 100571

Generation and validation of APOE knockout human iPSC-derived cerebral organoids.

Yuka A Martens, Siming Xu, Richard Tait, Gary Li, Xinping C Zhao, Wenyan Lu, Chia-Chen Liu, Takahisa Kanekiyo, Guojun Bu, Jing Zhao.

  Apolipoprotein E (apoE) is a major lipid carrier in the brain and closely associated with the pathogenesis of Alzheimer's disease (AD). Here, we describe a protocol for efficient knockout of APOE in human induced pluripotent stem cells (iPSCs) using the CRISPR-Cas9 system. We obtain homozygous APOE knockout (APOE -/- ) iPSCs and further validate the deficiency of apoE in iPSC-derived cerebral organoids. APOE -/- cerebral organoids can serve as a useful tool to study apoE functions and apoE-related pathogenic mechanisms in AD. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Keywords:  CRISPR; Neuroscience; Organoids; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2021.100571