bims-proarb 2022-02-20 papers

bims-proarb

Biomed News

on Proteostasis in aging and regenerative biology

Issue of 2022–02–20
27 papers selected by
Rich Giadone, Harvard University

Emerging fluorescence tools for the study of proteostasis in cells.
Aging alters the metabolic flux signature of the ER-unfolded protein response in vivo in mice.
Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain.
Circadian remodeling of the proteome by chaperone-mediated autophagy.
Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation.
Autophagy system as a potential therapeutic target for neurodegenerative diseases.
ATP-Independent Chaperones.
Deciphering the prion-like behavior of pathogenic protein aggregates in neurodegenerative diseases.
Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae.
Actions of Klotho on hippocampal neuronal cells.
Defective protein degradation in genetic disorders.
Aged Cattle Brain Displays Alzheimer's Disease-Like Pathology and Promotes Brain Amyloidosis in a Transgenic Animal Model.
Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.
Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L.
Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.
Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis.
XBP1u Is Involved in C2C12 Myoblast Differentiation via Accelerated Proteasomal Degradation of Id3.
Tau liquid-liquid phase separation in neurodegenerative diseases.
Deubiquitinases in Neurodegeneration.
Lifespan Increase of Podospora anserina by Oleic Acid Is Linked to Alterations in Energy Metabolism, Membrane Trafficking and Autophagy.
ER Unfolded Protein Response in Liver In Vivo Is Characterized by Reduced, Not Increased, De Novo Lipogenesis and Cholesterol Synthesis Rates with Uptake of Fatty Acids from Adipose Tissue: Integrated Gene Expression, Translation Rates and Metabolic Fluxes.
A human brain vascular atlas reveals diverse mediators of Alzheimer's risk.
An Overview of Methods for Detecting eIF2α Phosphorylation and the Integrated Stress Response.
Advances in Proteomic and Metabolomic Profiling of Neurodegenerative Diseases.
Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells.
BACE1 Overexpression Reduces SH-SY5Y Cell Viability Through a Mechanism Distinct from Amyloid-β Peptide Accumulation: Beta Prime-Mediated Competitive Depletion of sAβPPα.
Picturing protein disaggregation.

Curr Opin Chem Biol. 2022 Feb 14. pii: S1367-5931(22)00001-1. [Epub ahead of print]67 102116

Emerging fluorescence tools for the study of proteostasis in cells.

Tze Cin Owyong, Yuning Hong.

  Understanding how cells maintain the functional proteome and respond to stress conditions is critical for deciphering molecular pathogenesis and developing treatments for conditions such as neurodegenerative diseases. Efforts towards finer quantification of cellular proteostasis machinery efficiency, phase transitions and local environment changes remain a priority. Herein, we describe recent developments in fluorescence-based strategy and methodology, building on the experimental toolkit, for the study of proteostasis (protein homeostasis) in cells. We hope this review can assist in bridging gaps between a multitude of research disciplines and promote interdisciplinary collaboration to address the crucial topic of proteostasis.

Keywords:  Fluorescence imaging; Fluorescent probes; Protein aggregation; Protein misfolding; Proteostasis

DOI:  https://doi.org/10.1016/j.cbpa.2022.102116
Aging Cell. 2022 Feb 16. e13558

Aging alters the metabolic flux signature of the ER-unfolded protein response in vivo in mice.

Catherine P Ward, Lucy Peng, Samuel Yuen, John Halstead, Hector Palacios, Edna Nyangau, Hussein Mohammed, Naveed Ziari, Mohamad Dandan, Ashley E Frakes, Holly K Gildea, Andrew Dillin, Marc K Hellerstein.

  Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPRER ) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a flux "signature" of the UPRER in vivo in mouse liver was developed by inducing ER stress with tunicamycin and measuring rates of both proteome-wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared with young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was seen only in the triglyceride fraction. These data indicate that aged mice have an exaggerated metabolic flux response to ER stress, which may indicate that aging renders the UPRER less effective in resolving proteotoxic stress.

Keywords:  aging; de novo lipogenesis; endoplasmic reticulum; proteome dynamics; proteomics; unfolded protein response

DOI:  https://doi.org/10.1111/acel.13558
Aging Cell. 2022 Feb 15. e13559

Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain.

Ananth R Srinivasan, Tracy T Tran, Nancy M Bonini.

  Aging is a risk factor for neurodegenerative disease, but precise mechanisms that influence this relationship are still under investigation. Work in Drosophila melanogaster identified the microRNA miR-34 as a modifier of aging and neurodegeneration in the brain. MiR-34 mutants present aspects of early aging, including reduced lifespan, neurodegeneration, and a buildup of the repressive histone mark H3K27me3. To better understand how miR-34 regulated pathways contribute to age-associated phenotypes in the brain, here we transcriptionally profiled the miR-34 mutant brain. This identified that genes associated with translation are dysregulated in the miR-34 mutant. The brains of these animals show increased translation activity, accumulation of protein aggregation markers, and altered autophagy activity. To determine if altered H3K27me3 was responsible for this proteostasis dysregulation, we studied the effects of increased H3K27me3 by mutating the histone demethylase Utx. Reduced Utx activity enhanced neurodegeneration and mimicked the protein accumulation seen in miR-34 mutant brains. However, unlike the miR-34 mutant, Utx mutant brains did not show similar altered autophagy or translation activity, suggesting that additional miR-34-targeted pathways are involved. Transcriptional analysis of predicted miR-34 targets identified Lst8, a subunit of Tor Complex 1 (TORC1), as a potential target. We confirmed that miR-34 regulates the 3' UTR of Lst8 and identified several additional predicted miR-34 targets that may be critical for maintaining proteostasis and brain health. Together, these results present novel understanding of the brain and the role of the conserved miRNA miR-34 in impacting proteostasis in the brain with age.

Keywords:   miR-34 ; aging; autophagy; neurodegeneration; proteostasis; translation

DOI:  https://doi.org/10.1111/acel.13559
Autophagy. 2022 Feb 15. 1-3

Circadian remodeling of the proteome by chaperone-mediated autophagy.

Susmita Kaushik, Yves R Juste, Ana Maria Cuervo.

  The circadian clock drives daily cycles of physiology and behavioral outputs to keep organisms in tune with the environment. Cyclic oscillations in levels of the clock proteins maintain circadian rhythmicity. In our recent work, we have discovered the interdependence of the circadian clock and chaperone-mediated autophagy (CMA), a selective form of lysosomal protein degradation. Central and peripheral degradation of core clock proteins by CMA (selective chronophagy) modulates circadian rhythm. Loss of CMA in vivo disrupts physiological circadian cycling, resembling defects observed in aging, a condition with reduced CMA. Conversely, the circadian clock temporally regulates CMA activity in a tissue-specific manner, contributing to remodeling of a distinct subproteome at different circadian times. This timely remodeling cannot be sustained when CMA fails, despite rerouting of some CMA substrates to other degradation pathways.

Keywords:  Central clock; chaperones; circadian rhythms; lysosomes; organelle proteomics; peripheral clock

DOI:  https://doi.org/10.1080/15548627.2022.2038503
Acta Neuropathol Commun. 2022 Feb 14. 10(1): 22

Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation.

Feilin Liu, Dmytro Morderer, Melissa C Wren, Sara A Vettleson-Trutza, Yanzhe Wang, Benjamin E Rabichow, Michelle R Salemi, Brett S Phinney, Björn Oskarsson, Dennis W Dickson, Wilfried Rossoll.

  The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.

Keywords:  C9orf72; Heat shock proteins; Poly-GA; Proximity proteomics

DOI:  https://doi.org/10.1186/s40478-022-01322-x
Neurochem Int. 2022 Feb 15. pii: S0197-0186(22)00033-X. [Epub ahead of print] 105308

Autophagy system as a potential therapeutic target for neurodegenerative diseases.

Mengying Cui, Tamotsu Yoshimori, Shuhei Nakamura.

  Autophagy is an evolutionally conserved process by which cytoplasmic contents including protein aggregates and damaged organelles such as mitochondria and lysosomes, are sequestered by double-membrane structure, autophagosomes, and delivered to the lysosomes for degradation. Recently, considerable efforts have been made to reveal the role of autophagy in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease. Impairment of autophagy aggravates the accumulation of misfolded protein and damaged organelles in neurons, while sufficient autophagic activity reduces such accumulation in nervous system and ameliorates the pathology. Here we summarize recent progress regarding the role of autophagy in several neurodegenerative diseases and the potential autophagy-associated therapies for them.

Keywords:  Aggregates; Autophagy; Neurodegenerative disease; Therapy

DOI:  https://doi.org/10.1016/j.neuint.2022.105308
Annu Rev Biophys. 2022 Feb 15.

ATP-Independent Chaperones.

Rishav Mitra, Kevin Wu, Changhan Lee, James C A Bardwell.

The folding of proteins into their native structure is crucial for the functioning of all biological processes. Molecular chaperones are guardians of the proteome that assist in protein folding and prevent the accumulation of aberrant protein conformations that can lead to proteotoxicity. ATP-independent chaperones do not require ATP to regulate their functional cycle. Although these chaperones have been traditionally regarded as passive holdases that merely prevent aggregation, recent work has shown that they can directly affect the folding energy landscape by tuning their affinity to various folding states of the client. This review focuses on emerging paradigms in the mechanism of action of ATP-independent chaperones and on the various modes of regulating client binding and release. 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-090121-082906
Neurochem Int. 2022 Feb 15. pii: S0197-0186(22)00032-8. [Epub ahead of print] 105307

Deciphering the prion-like behavior of pathogenic protein aggregates in neurodegenerative diseases.

Shun Yoshida, Takafumi Hasegawa.

  Neurodegenerative diseases are hitherto classified based on their core clinical features, the anatomical distribution of neurodegeneration, and the cell populations mainly affected. On the other hand, the wealth of neuropathological, genetic, molecular and biochemical studies have identified the existence of distinct insoluble protein aggregates in the affected brain regions. These findings have spread the use of a collective term, proteinopathy, for neurodegenerative disorders with particular type of structurally altered protein accumulation. Particularly, a recent breakthrough in this field came with the discovery that these protein aggregates can transfer from one cell to another, thereby converting normal proteins to potentially toxic, misfolded species in a prion-like manner. In this review, we focus specifically on the molecular and cellular basis that underlies the seeding activity and transcellular spreading phenomenon of neurodegeneration-related protein aggregates, and discuss how these events contribute to the disease progression.

Keywords:  Alzheimer's disease; Amyotrophic lateral sclerosis; Huntington's disease; Parkinson's disease; Prion-like transmission; Protein aggregates

DOI:  https://doi.org/10.1016/j.neuint.2022.105307
Int J Mol Sci. 2022 Jan 31. pii: 1669. [Epub ahead of print]23(3):

Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae.

Tatsuya Hata, Yuki Ishiwata-Kimata, Yukio Kimata.

  Ire1 is an endoplasmic reticulum (ER)-located endoribonuclease that is activated in response to ER stress. In yeast Saccharomyces cerevisiae cells, Ire1 promotes HAC1-mRNA splicing to remove the intron sequence from the HAC1u mRNA ("u" stands for "uninduced"). The resulting mRNA, which is named HAC1i mRNA ("i" stands for "induced"), is then translated into a transcription factor that is involved in the unfolded protein response (UPR). In this study, we designed an oligonucleotide primer that specifically hybridizes to the exon-joint site of the HAC1i cDNA. This primer allowed us to perform real-time reverse transcription-PCR to quantify HAC1i mRNA abundance with high sensitivity. Using this method, we detected a minor induction of HAC1-mRNA splicing in yeast cells cultured at their maximum growth temperature of 39 °C. Based on our analyses of IRE1-gene mutant strains, we propose that when yeast cells are cultured at or near their maximum growth temperature, protein folding in the ER is disturbed, leading to a minor UPR induction that supports cellular growth.

Keywords:  PCR; endoplasmic reticulum; mRNA solicing; yeast

DOI:  https://doi.org/10.3390/ijms23031669
Vitam Horm. 2022 ;pii: S0083-6729(21)00078-9. [Epub ahead of print]118 223-246

Actions of Klotho on hippocampal neuronal cells.

Jennifer Mytych.

  Klotho gene was originally recognized as a putative aging-suppressor and its prominent age-regulating effects are mostly attributed to the modulation of mineral homeostasis in the kidney. However, recent studies link alterations in hippocampal Klotho expression with cognitive impairment and neurodegenerative diseases. This suggests that hippocampal neurons require Klotho for health and proper functionality. Klotho protects against neuronal dysfunction and regulates several intracellular signaling pathways including oxidative stress response, inflammation, DNA damage, autophagy, endoplasmic reticulum stress response, and multiple types of cell death. Specifically, this chapter covers the current knowledge as to how Klotho protein affects the hippocampal neuronal cells, with special attention paid to underlying molecular mechanisms, and thus influences hippocampal development, hippocampal-dependent cognition, behavior, and motor skills as well as mediates neurodegenerative processes.

Keywords:  Autophagy; Cell death; DNA damage; ER stress; Hippocampus; Inflammation; Klotho; Neurons; Oxidative stress

DOI:  https://doi.org/10.1016/bs.vh.2021.12.001
Biochim Biophys Acta Mol Basis Dis. 2022 Feb 11. pii: S0925-4439(22)00029-1. [Epub ahead of print] 166366

Defective protein degradation in genetic disorders.

Pau Castel.

  Understanding the molecular mechanisms that underlie different human pathologies is necessary to develop novel therapeutic strategies. An emerging mechanism of pathogenesis in many genetic disorders is the dysregulation of protein degradation, which leads to the accumulation of proteins that are responsible for the disease phenotype. Among the different cellular pathways that regulate active proteolysis, the Cullin RING E3 ligases represent an important group of sophisticated enzymatic complexes that mediate substrate ubiquitination through the interaction with specific adaptors. However, pathogenic mutations in these adaptors affect the physiological ubiquitination of their substrates. This review discusses our current understanding of this emerging field.

Keywords:  BTB proteins; CRL3; Congenital disorders; Cullin 3; LZTR1; Ubiquitin

DOI:  https://doi.org/10.1016/j.bbadis.2022.166366
Front Aging Neurosci. 2021 ;13 815361

Aged Cattle Brain Displays Alzheimer's Disease-Like Pathology and Promotes Brain Amyloidosis in a Transgenic Animal Model.

Ines Moreno-Gonzalez, George Edwards, Rodrigo Morales, Claudia Duran-Aniotz, Gabriel Escobedo, Mercedes Marquez, Marti Pumarola, Claudio Soto.

  Alzheimer's disease (AD) is one of the leading causes of dementia in late life. Although the cause of AD neurodegenerative changes is not fully understood, extensive evidence suggests that the misfolding, aggregation and cerebral accumulation of amyloid beta (Aβ) and tau proteins are hallmark events. Recent reports have shown that protein misfolding and aggregation can be induced by administration of small quantities of preformed aggregates, following a similar principle by which prion diseases can be transmitted by infection. In the past few years, many of the typical properties that characterize prions as infectious agents were also shown in Aβ aggregates. Interestingly, prion diseases affect not only humans, but also various species of mammals, and it has been demonstrated that infectious prions present in animal tissues, particularly cattle affected by bovine spongiform encephalopathy (BSE), can infect humans. It has been reported that protein deposits resembling Aβ amyloid plaques are present in the brain of several aged non-human mammals, including monkeys, bears, dogs, and cheetahs. In this study, we investigated the presence of Aβ aggregates in the brain of aged cattle, their similarities with the protein deposits observed in AD patients, and their capability to promote AD pathological features when intracerebrally inoculated into transgenic animal models of AD. Our data show that aged cattle can develop AD-like neuropathological abnormalities, including amyloid plaques, as studied histologically. Importantly, cow-derived aggregates accelerate Aβ amyloid deposition in the brain of AD transgenic animals. Surprisingly, the rate of induction produced by administration of the cattle material was substantially higher than induction produced by injection of similar amounts of human AD material. Our findings demonstrate that cows develop seeding-competent Aβ aggregates, similarly as observed in AD patients.

Keywords:  Alzheimer's disease; amyloid; cattle; prions; protein misfolding; seeding; spreading

DOI:  https://doi.org/10.3389/fnagi.2021.815361
Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2102248119. [Epub ahead of print]119(8):

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.

Yong Bhum Song, Seung-Yeol Park, Kunyou Park, Hayoung Hwang, Rona S Carroll, Victor W Hsu, Ursula B Kaiser.

  G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

Keywords:  Golgi; PROKR2; endoplasmic reticulum

DOI:  https://doi.org/10.1073/pnas.2102248119
Cell Rep. 2022 02 15. pii: S2211-1247(22)00091-2. [Epub ahead of print]38(7): 110370

Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L.

Gulzar A Wani, Hans-Georg Sprenger, Kristiano Ndoci, Srikanth Chandragiri, Richard James Acton, Désirée Schatton, Sandra M V Kochan, Vignesh Sakthivelu, Milica Jevtic, Jens M Seeger, Stefan Müller, Patrick Giavalisco, Elena I Rugarli, Elisa Motori, Thomas Langer, Matteo Bergami.

  The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled with reversible changes in energy metabolism with key implications for lifelong NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We find that a state-specific rewiring of the mitochondrial proteome by the i-AAA peptidase YME1L is required to preserve NSPC self-renewal. YME1L controls the abundance of numerous mitochondrial substrates in quiescent NSPCs, and its deletion activates a differentiation program characterized by broad metabolic changes causing the irreversible shift away from a fatty-acid-oxidation-dependent state. Conditional Yme1l deletion in adult NSPCs in vivo results in defective self-renewal and premature differentiation, ultimately leading to NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.

Keywords:  OMA1; YME1L; adult neurogenesis; metabolic rewiring; mitochondria; mitochondrial dynamics; mitochondrial proteome; neural stem cells; proliferation; self-renewal

DOI:  https://doi.org/10.1016/j.celrep.2022.110370
Nat Chem Biol. 2022 Feb 17.

Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.

Yoshiko Nakagawa, Howard C-H Shen, Yusuke Komi, Shinju Sugiyama, Takaaki Kurinomaru, Yuri Tomabechi, Elena Krayukhina, Kenji Okamoto, Takeshi Yokoyama, Mikako Shirouzu, Susumu Uchiyama, Megumi Inaba, Tatsuya Niwa, Yasushi Sako, Hideki Taguchi, Motomasa Tanaka.

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41589-021-00951-y
EMBO J. 2022 Feb 16. e109845

Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis.

John C Christianson, Pedro Carvalho.

  The endoplasmic reticulum (ER) is a large, dynamic, and multifunctional organelle. ER protein homeostasis is essential for the coordination of its diverse functions and depends on ER-associated protein degradation (ERAD). The latter process selects target proteins in the lumen and membrane of the ER, promotes their ubiquitination, and facilitates their delivery into the cytosol for degradation by the proteasome. Originally characterized for a role in the degradation of misfolded proteins and rate-limiting enzymes of sterol biosynthesis, the many branches of ERAD now appear to control the levels of a wider range of substrates and influence more broadly the organization and functions of the ER, as well as its interactions with adjacent organelles. Here, we discuss recent mechanistic advances in our understanding of ERAD and of its consequences for the regulation of ER functions.

Keywords:  ERAD; endoplasmic reticulum; protein degradation; protein quality control; ubiquitin ligase

DOI:  https://doi.org/10.15252/embj.2021109845
Front Physiol. 2022 ;13 796190

XBP1u Is Involved in C2C12 Myoblast Differentiation via Accelerated Proteasomal Degradation of Id3.

Satoko Hayashi, Shotaro Sakata, Shotaro Kawamura, Yukako Tokutake, Shinichi Yonekura.

  Myoblast differentiation is an ordered multistep process that includes withdrawal from the cell cycle, elongation, and fusion to form multinucleated myotubes. Id3, a member of the Id family, plays a crucial role in cell cycle exit and differentiation. However, in muscle cells after differentiation induction, the detailed mechanisms that diminish Id3 function and cause the cells to withdraw from the cell cycle are unknown. Induction of myoblast differentiation resulted in decreased expression of Id3 and increased expression of XBP1u, and XBP1u accelerated proteasomal degradation of Id3 in C2C12 cells. The expression levels of the cyclin-dependent kinase inhibitors p21, p27, and p57 were not increased after differentiation induction of XBP1-knockdown C2C12 cells. Moreover, knockdown of Id3 rescued myogenic differentiation of XBP1-knockdown C2C12 cells. Taken together, these findings provide evidence that XBP1u regulates cell cycle exit after myogenic differentiation induction through interactions with Id3. To the best of our knowledge, this is the first report of the involvement of XBP1u in myoblast differentiation. These results indicate that XBP1u may act as a "regulator" of myoblast differentiation under various physiological conditions.

Keywords:  Id3; cell cycle exit; cyclin-dependent kinase inhibitor; skeletal muscle differentiation; unfolded protein response

DOI:  https://doi.org/10.3389/fphys.2022.796190
Trends Cell Biol. 2022 Feb 15. pii: S0962-8924(22)00026-5. [Epub ahead of print]

Tau liquid-liquid phase separation in neurodegenerative diseases.

Solomiia Boyko, Witold K Surewicz.

  Aggregation of the microtubule-associated protein tau plays a major role in Alzheimer's disease and several other neurodegenerative disorders. An exciting recent development is the finding that, akin to some other proteins associated with neurodegenerative disease, tau has a high propensity to condensate via the mechanism of liquid-liquid phase separation (LLPS). Here, we discuss the evidence for tau LLPS in vitro, the molecular mechanisms of this reaction, and the role of post-translational modifications and pathogenic mutations in tau phase separation. We also discuss recent studies on tau LLPS in cells and the insights these studies provide regarding the link between LLPS and neurodegeneration in tauopathies.

Keywords:  liquid–liquid phase separation; neurodegenerative diseases; protein aggregation; protein condensation; tau

DOI:  https://doi.org/10.1016/j.tcb.2022.01.011
Cells. 2022 Feb 05. pii: 556. [Epub ahead of print]11(3):

Deubiquitinases in Neurodegeneration.

Abudu I Bello, Rituparna Goswami, Shelby L Brown, Kara Costanzo, Taylor Shores, Shefaa Allan, Revan Odah, Ryan D Mohan.

  Ubiquitination refers to the conjugation of the ubiquitin protein (a small protein highly conserved among eukaryotes) to itself or to other proteins through differential use of ubiquitin's seven internal linkage sites or the amino-terminal amino group. By creating different chain lengths, an enormous proteomic diversity may be formed. This creates a signaling system that is central to controlling almost every conceivable protein function, from proteostasis to regulating enzyme function and everything in between. Protein ubiquitination is reversed through the activity of deubiquitinases (DUBs), enzymes that function to deconjugate ubiquitin from itself and protein substrates. DUBs are regulated through several mechanisms, from controlled subcellular localization within cells to developmental and tissue specific expression. Misregulation of DUBs has been implicated in several diseases including cancer and neurodegeneration. Here we present a brief overview of the role of DUBs in neurodegeneration, and as potential therapeutic targets.

Keywords:  Drosophila; deubiquitinase; neurodegeneration; ubiquitin; ubiquitin-specific protease

DOI:  https://doi.org/10.3390/cells11030556
Cells. 2022 Feb 02. pii: 519. [Epub ahead of print]11(3):

Lifespan Increase of Podospora anserina by Oleic Acid Is Linked to Alterations in Energy Metabolism, Membrane Trafficking and Autophagy.

Lea Schürmanns, Andrea Hamann, Heinz D Osiewacz.

  The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.

Keywords:  ATG24; ER; Podospora anserina; aging; autophagy; membrane trafficking; mitochondria; peroxisomes

DOI:  https://doi.org/10.3390/cells11030519
Int J Mol Sci. 2022 Jan 19. pii: 1073. [Epub ahead of print]23(3):

ER Unfolded Protein Response in Liver In Vivo Is Characterized by Reduced, Not Increased, De Novo Lipogenesis and Cholesterol Synthesis Rates with Uptake of Fatty Acids from Adipose Tissue: Integrated Gene Expression, Translation Rates and Metabolic Fluxes.

Catherine P Ward, Lucy Peng, Samuel Yuen, Michael Chang, Rozalina Karapetyan, Edna Nyangau, Hussein Mohammed, Hector Palacios, Naveed Ziari, Larry K Joe, Ashley E Frakes, Mohamad Dandan, Andrew Dillin, Marc K Hellerstein.

  The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases. Here, we characterize UPRER-induced metabolic changes in mouse livers in vivo through metabolic labeling and mass spectrometric analysis of lipid and proteome-wide fluxes. We induced UPRER by tunicamycin administration and measured synthesis rates of proteins, fatty acids and cholesterol, as well as RNA-seq. Contrary to reports in isolated cells, hepatic de novo lipogenesis and cholesterogenesis were markedly reduced, as were mRNA levels and synthesis rates of lipogenic proteins. H&E staining showed enrichment with lipid droplets while electron microscopy revealed ER morphological changes. Interestingly, the pre-labeling of adipose tissue prior to UPRER induction resulted in the redistribution of labeled fatty acids from adipose tissue to the liver, with replacement by unlabeled glycerol in the liver acylglycerides, indicating that the liver uptake was of free fatty acids, not whole glycerolipids. The redistribution of adipose fatty acids to the liver was not explicable by altered plasma insulin, increased fatty acid levels (lipolysis) or by reduced food intake. Synthesis of most liver proteins was suppressed under UPRER conditions, with the exception of BiP, other chaperones, protein disulfide isomerases, and proteins of ribosomal biogenesis. Protein synthesis rates generally, but not always, paralleled changes in mRNA. In summary, this combined approach, linking static changes with fluxes, revealed an integrated reduction of lipid and cholesterol synthesis pathways, from gene expression to translation and metabolic flux rates, under UPRER conditions. The reduced lipogenesis does not parallel human fatty liver disease. This approach provides powerful tools to characterize metabolic processes underlying hepatic UPRER in vivo.

Keywords:  ER stress; UPR; lipid metabolism; metabolism; proteostasis

DOI:  https://doi.org/10.3390/ijms23031073
Nature. 2022 Feb 14.

A human brain vascular atlas reveals diverse mediators of Alzheimer's risk.

Andrew C Yang, Ryan T Vest, Fabian Kern, Davis P Lee, Maayan Agam, Christina A Maat, Patricia M Losada, Michelle B Chen, Nicholas Schaum, Nathalie Khoury, Angus Toland, Kruti Calcuttawala, Heather Shin, Róbert Pálovics, Andrew Shin, Elizabeth Y Wang, Jian Luo, David Gate, Walter J Schulz-Schaeffer, Pauline Chu, Julie A Siegenthaler, M Windy McNerney, Andreas Keller, Tony Wyss-Coray.

The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.

DOI: https://doi.org/10.1038/s41586-021-04369-3
Methods Mol Biol. 2022 ;2428 3-18

An Overview of Methods for Detecting eIF2α Phosphorylation and the Integrated Stress Response.

Agnieszka Krzyzosiak, Aleksandra P Pitera, Anne Bertolotti.

  Phosphorylation of the translation initiation factor eIF2α is an adaptive signaling event that is essential for cell and organismal survival from yeast to humans. It is central to the integrated stress response (ISR) that maintains cellular homeostasis in the face of threats ranging from viral infection, amino acid, oxygen, and heme deprivation to the accumulation of misfolded proteins in the endoplasmic reticulum. Phosphorylation of eIF2α has broad physiological, pathological, and therapeutic relevance. However, despite more than two decades of research and growing pharmacological interest, phosphorylation of eIF2α remains difficult to detect and quantify, because of its transient nature and because substoichiometric amounts of this modification are sufficient to profoundly reshape cellular physiology. This review aims to provide a roadmap for facilitating a robust evaluation of eIF2α phosphorylation and its downstream consequences in cells and organisms.

Keywords:  ATF4; CHOP; Integrated stress response; PPP1R15A/GADD34; PPP1R15B/CReP; Signaling; Stress signaling; Translation; Unfolded protein response; eIF2α dephosphorylation; eIF2α phosphorylation

DOI:  https://doi.org/10.1007/978-1-0716-1975-9_1
Front Neurol. 2021 ;12 792227

Advances in Proteomic and Metabolomic Profiling of Neurodegenerative Diseases.

Artur Schumacher-Schuh, Andrei Bieger, Wyllians V Borelli, Makayla K Portley, Paula Saffie Awad, Sara Bandres-Ciga.

  Proteomics and metabolomics are two emerging fields that hold promise to shine light on the molecular mechanisms causing neurodegenerative diseases. Research in this area may reveal and quantify specific metabolites and proteins that can be targeted by therapeutic interventions intended at halting or reversing the neurodegenerative process. This review aims at providing a general overview on the current status of proteomic and metabolomic profiling in neurodegenerative diseases. We focus on the most common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. We discuss the relevance of state-of-the-art metabolomics and proteomics approaches and their potential for biomarker discovery. We critically review advancements made so far, highlighting how metabolomics and proteomics may have a significant impact in future therapeutic and biomarker development. Finally, we further outline technologies used so far as well as challenges and limitations, placing the current information in a future-facing context.

Keywords:  AD; ALS; Alzheimer's disease; PD; Parkinson's disease; amyotrophic lateral sclerosis; metabolomics; proteomics

DOI:  https://doi.org/10.3389/fneur.2021.792227
Cell Death Discov. 2022 Feb 17. 8(1): 69

Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells.

Jing Lv, Ying Yi, Yan Qi, Chenchao Yan, Wenwen Jin, Liming Meng, Donghui Zhang, Wei Jiang.

Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

DOI: https://doi.org/10.1038/s41420-022-00867-z
J Alzheimers Dis. 2022 Feb 15.

BACE1 Overexpression Reduces SH-SY5Y Cell Viability Through a Mechanism Distinct from Amyloid-β Peptide Accumulation: Beta Prime-Mediated Competitive Depletion of sAβPPα.

Lauren Owens, Joshua Bracewell, Alexandre Benedetto, Neil Dawson, Christopher Gaffney, Edward Parkin.

   BACKGROUND: The Alzheimer's disease (AD)-associated amyloid-beta protein precursor (AβPP) can be cleaved by β-site AβPP cleaving enzyme 1 (BACE1) and the γ-secretase complex to yield neurotoxic amyloid-β (Aβ) peptides. However, AβPP can also be cleaved in a 'non-amyloidogenic' manner either by α-secretase to produce soluble AβPP alpha (sAβPPα) (a fragment with neuroprotective/neurogenic functions) or through alternative BACE1-mediated 'beta prime' activity yielding soluble AβPP beta prime (sAβPPβ').
OBJECTIVE: To determine whether sAβPPα depletion, as opposed to Aβ peptide accumulation, contributes to cytotoxicity in AD-relevant SH-SY5Y neuroblastoma cell models.
METHODS: AβPP proteolysis was characterized by immunoblotting in mock-, wild-type AβPP (wtAβPP)-, BACE1-, and Swedish mutant AβPP (SweAβPP)-transfected cells. AβPP beta prime cleavage was confirmed through secretase inhibitor studies and C-terminal fragment analysis. The roles of sAβPPα and sAβPPβ' in cell viability were confirmed by overexpression studies.
RESULTS: Despite producing enhanced Aβ peptide levels, wtAβPP- and SweAβPP-transfected cells did not exhibit reduced viability whereas BACE1-transfected cells did. sAβPPα generation in SH-SY5Y-BACE1 cells was virtually ablated in lieu of BACE1-mediated sAβPPβ' production. sAβPPα overexpression in SH-SY5Y-BACE1 cells restored viability whereas sAβPPβ' overexpression decreased viability further. The anti-AβPP 6E10 antibody was shown to cross-react with sAβPPβ'.
CONCLUSION: sAβPPα depletion and/or sAβPPβ' accumulation, but not elevated Aβ peptide levels, represent the cytotoxic mechanism following BACE1 overexpression in SH-SY5Y cells. These data support the novel concept that competitive sAβPPα depletion by BACE1 beta prime activity might contribute to AD. The cross-reactivity of 6E10 with AβPPβ'also questions whether previous studies assessing sAβPPα as a biomarker using this antibody should be revisited.

Keywords:  Alpha-secretase; Alzheimer’s disease; amyloid-beta protein precursor; beta prime; beta-secretase

DOI:  https://doi.org/10.3233/JAD-215457
Nat Chem Biol. 2022 Feb 17.

Picturing protein disaggregation.

Sander J Tans.

DOI: https://doi.org/10.1038/s41589-022-00977-w