Granulins modulate liquid-liquid phase separation and aggregation of TDP-43 C-terminal domain

ABSTRACTTar DNA binding protein (TDP-43) has emerged as a key player in many neurodegenerative pathologies including frontotemporal lobar degeneration (FTLD) and amyotropic lateral sclerosis (ALS). Important hallmarks of FTLD and ALS are the toxic cytoplasmic inclusions of C-terminal fragments of TDP-43 (TDP-43CTD), which are formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. TDP-43CTD is also known to form stress granules (SGs) by coacervating with RNA in cytoplasm under stress conditions and are believed to be involved in modulating the pathologies. Among other factors affecting these pathologies, the pleiotropic protein called progranulin (PGRN) has gained significant attention lately. The haploinsufficiency of PGRN, caused by autosomal dominant mutations in GRN gene, results in its loss-of-function linked to FTLD and ALS. But precisely how the protein contributes to the pathology remains unknown. Recently, cleavage to GRNs were observed to be a significant part of FTLD and ALS progression with specific GRNs exacerbating TDP-43-induced toxicity in C.elegans. In this report, we show that GRNs −3 and −5 directly interact with TDP-43CTD to modulate latter’s aggregation or stress granule formation in disparate ways in vitro. These results constitute the first observation of direct interaction between GRNs and TDP-43 and suggest a mechanism by which the loss of PGRN function could lead to FTLD and ALS.

TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD

10.1101/2020.09.15.298455 ◽

2020 ◽

Author(s):

Hadjara Sidibé ◽

Yousra Khalfallah ◽

Shangxi Xiao ◽

Nicolás B. Gómez ◽

Elizabeth M.H. Tank ◽

...

Keyword(s):

Regulatory Element ◽

Stress Granule ◽

Loss Of Function ◽

Cytoplasmic Inclusions ◽

Protein Levels ◽

Assembly Factor ◽

Direct Binding ◽

ABSTRACTTDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3’UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013728 ◽

2020 ◽

Vol 295 (39) ◽

pp. 13617-13629

Author(s):

Clément Immarigeon ◽

Sandra Bernat-Fabre ◽

Emmanuelle Guillou ◽

Alexis Verger ◽

Elodie Prince ◽

...

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Direct Interaction ◽

Mediator Complex ◽

Loss Of Function ◽

Transcriptional Responses ◽

Rna Pol Ii ◽

Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

10.1101/721001 ◽

2019 ◽

Cited By ~ 8

Author(s):

Richard J. Wheeler ◽

Hyun O. Lee ◽

Ina Poser ◽

Arun Pal ◽

Thom Doeleman ◽

...

Keyword(s):

Phase Separation ◽

Neurodegenerative Disease ◽

Stress Granules ◽

Life Time ◽

Stress Granule ◽

Phase Behaviour ◽

Granule Proteins ◽

Lateral Sclerosis ◽

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with few avenues for treatment. Many proteins implicated in ALS associate with stress granules, which are examples of liquid-like compartments formed by phase separation. Aberrant phase transition of stress granules has been implicated in disease, suggesting that modulation of phase transitions could be a possible therapeutic route. Here, we combine cell-based and protein-based screens to show that lipoamide, and its related compound lipoic acid, reduce the propensity of stress granule proteins to aggregate in vitro. More significantly, they also prevented aggregation of proteins over the life time of Caenorhabditis elegans. Observations that they prevent dieback of ALS patient-derived (FUS mutant) motor neuron axons in culture and recover motor defects in Drosophila melanogaster expressing FUS mutants suggest plausibility as effective therapeutics. Our results suggest that altering phase behaviour of stress granule proteins in the cytoplasm could be a novel route to treat ALS.

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

The Journal of Cell Biology ◽

10.1083/jcb.202103160 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Chen Chen ◽

Yoshiaki Yamanaka ◽

Koji Ueda ◽

Peiying Li ◽

Tamami Miyagi ◽

...

Keyword(s):

Phase Separation ◽

Charge Distribution ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C9orf72 Gene ◽

And Diffusion ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽

10.3390/cells9081791 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1791

Author(s):

Ana Bajc Česnik ◽

Helena Motaln ◽

Boris Rogelj

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Low Complexity ◽

Wild Type ◽

Disease Heterogeneity ◽

Cytoplasmic Inclusions ◽

The Impact ◽

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice

Cells ◽

10.3390/cells9112390 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2390

Author(s):

Ankush Borlepawar ◽

Nesrin Schmiedel ◽

Matthias Eden ◽

Lynn Christen ◽

Alexandra Rosskopf ◽

...

Keyword(s):

Direct Interaction ◽

Cardiomyocyte Hypertrophy ◽

Loss Of Function ◽

Interaction Partners ◽

Lysosome Related Organelles ◽

The Stability ◽

Schizophrenia Susceptibility

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5–7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin’s role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

Granulins modulate liquid–liquid phase separation and aggregation of the prion-like C-terminal domain of the neurodegeneration-associated protein TDP-43

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011501 ◽

2020 ◽

Vol 295 (8) ◽

pp. 2506-2519 ◽

Cited By ~ 4

Author(s):

Anukool A. Bhopatkar ◽

Vladimir N. Uversky ◽

Vijayaraghavan Rangachari

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Structural Disorder ◽

Liquid Phase Separation ◽

Proteolytic Processing ◽

Full Length ◽

Cytoplasmic Inclusions ◽

Terminal Domain ◽

TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hallmarks of both FTLD and ALS are the toxic cytoplasmic inclusions of the prion-like C-terminal fragments of TDP-43 CTD (TDP-43 C-terminal domain), formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. Both full-length TDP-43 and its CTD are also known to form stress granules by coacervating with RNA in the cytoplasm during stress and may be involved in these pathologies. Furthermore, mutations in the PGRN gene, leading to haploinsufficiency and diminished function of progranulin (PGRN) protein, are strongly linked to FTLD and ALS. Recent reports have indicated that proteolytic processing of PGRN to smaller protein modules called granulins (GRNs) contributes to FTLD and ALS progression, with specific GRNs exacerbating TDP-43–induced cytotoxicity. Here we investigated the interactions between the proteolytic products of both TDP-43 and PGRN. Based on structural disorder and charge distributions, we hypothesized that GRN-3 and GRN-5 could interact with the TDP-43 CTD. We show that, under both reducing and oxidizing conditions, GRN-3 and GRN-5 interact with and differentially modulate TDP-43 CTD aggregation and/or liquid–liquid phase separation in vitro. GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid–liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS.

Plants-Derived Neuroprotective Agents: Cutting the Cycle of Cell Death through Multiple Mechanisms

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/3574012 ◽

2017 ◽

Vol 2017 ◽

pp. 1-27 ◽

Cited By ~ 17

Author(s):

Taiwo Olayemi Elufioye ◽

Tomayo Ireti Berida ◽

Solomon Habtemariam

Keyword(s):

Mechanisms Of Action ◽

Neuroprotective Agents ◽

Structural Class ◽

Molecular Features ◽

Amyotropic Lateral Sclerosis ◽

Underlying Mechanisms ◽

And Function ◽

Neuroprotection is the preservation of the structure and function of neurons from insults arising from cellular injuries induced by a variety of agents or neurodegenerative diseases (NDs). The various NDs including Alzheimer’s, Parkinson’s, and Huntington’s diseases as well as amyotropic lateral sclerosis affect millions of people around the world with the main risk factor being advancing age. Each of these diseases affects specific neurons and/or regions in the brain and involves characteristic pathological and molecular features. Hence, several in vitro and in vivo study models specific to each disease have been employed to study NDs with the aim of understanding their underlying mechanisms and identifying new therapeutic strategies. Of the most prevalent drug development efforts employed in the past few decades, mechanisms implicated in the accumulation of protein-based deposits, oxidative stress, neuroinflammation, and certain neurotransmitter deficits such as acetylcholine and dopamine have been scrutinized in great detail. In this review, we presented classical examples of plant-derived neuroprotective agents by highlighting their structural class and specific mechanisms of action. Many of these natural products that have shown therapeutic efficacies appear to be working through the above-mentioned key multiple mechanisms of action.

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

10.1101/402404 ◽

2018 ◽

Author(s):

Ruchika Sachdev ◽

Maria Hondele ◽

Miriam Linsenmeier ◽

Pascal Vallotton ◽

Christopher F. Mugler ◽

...

Keyword(s):

Phase Separation ◽

Direct Interaction ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

The Dead ◽

Processing Body ◽

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

Structural basis for mutation-induced destabilization of profilin 1 in ALS

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424108112 ◽

2015 ◽

Vol 112 (26) ◽

pp. 7984-7989 ◽

Cited By ~ 42

Author(s):

Sivakumar Boopathy ◽

Tania V. Silvas ◽

Maeve Tischbein ◽

Silvia Jansen ◽

Shivender M. Shandilya ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Structural Basis ◽

Loss Of Function ◽

Native Conformation ◽

Fatal Disease ◽

X Ray ◽

High Propensity ◽

Pathological Mechanism ◽

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.