Frameshift peptides alter the properties of truncated FUS proteins in ALS-FUS

Mutations in the FUS gene cause a subset of ALS cases (ALS-FUS). The majority of FUS mutations are missense mutations affecting the nuclear localisation signal (NLS) of FUS. In addition, a number of frameshift mutations which result in complete NLS deletion have been described. Patients bearing frameshift mutations usually present with more aggressive disease, characterised by an early onset and rapid progression. Both missense mutations in the NLS coding sequence and complete loss of the NLS are known to result in cytoplasmic mislocalisation of FUS protein. However, in addition to the removal of FUS functional domains, frameshift mutations in most cases lead to the attachment of a “tail” of novel amino acids at the FUS C-terminus – a frameshift peptide. It is not clear whether these peptide tails would affect the properties of truncated FUS proteins. In the current study, we compared intracellular behaviour of disease-associated truncated FUS proteins with and without the corresponding frameshift peptides. We demonstrate that some of these peptides can affect subcellular distribution and/or increase aggregation capacity and stability of the truncated FUS protein. Our study suggests that frameshift peptides can alter the properties of truncated FUS variants which may modulate FUS pathogenicity and contribute to the variability of the disease course in ALS-FUS

Connecting the "dots": RNP granule network in health and disease

All cells contain ribonucleoprotein (RNP) granules – large membraneless structures composed of RNA and proteins. Recent breakthroughs in RNP granule research have brought a new appreciation of their crucial role in organising virtually all cellular processes. Cells widely exploit the flexible, dynamic nature of RNP granules to adapt to a variety of functional states and the ever-changing environment. Constant exchange of molecules between the different RNP granules connects them into a network. This network controls basal cellular activities and is remodelled to enable efficient stress response. Alterations in RNP granule structure and regulation have been found to lead to fatal human diseases. The interconnectedness of RNP granules suggests that the RNP granule network as a whole becomes affected in disease states such as a representative neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this review, we summarize available evidence on the communication between different RNP granules and on the RNP granule network disruption as a primary ALS pathomechanism

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function

Aims The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. Methods and Results We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively-charged side chains of two aspartate residues (D179 and D180) within the β8-β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8-β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8-β9 domain in channel closing. Conclusions These results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression. Translational Potential Our findings that the RyR2 β8-β9 loop is involved in both Ca2+ release channel opening and closing have important clinical implications. This RyR2 domain is a known “hot-spot” for mutations associated with arrhythmogenic cardiac disease, which could produce hypersensitive as well as hyposensitive channels. Therapeutic strategies currently focus on gain-of-function RyR2 channels to suppress sarcoplasmic reticulum Ca2+ release either indirectly with class I/II anti-arrhythmic drugs, or by directly targeting RyR2 to inhibit channel activity. These strategies may not only be ineffective, but they may exacerbate the malignant phenotype in the case of loss-of-function RyR2 mutations

Frameshift peptides alter the properties of truncated FUS proteins in ALS-FUS

Mutations in the FUS gene cause a subset of ALS cases (ALS-FUS). The majority of FUS mutations are missense mutations affecting the nuclear localisation signal (NLS) of FUS. In addition, a number of frameshift mutations which result in complete NLS deletion have been described. Patients bearing frameshift mutations usually present with more aggressive disease, characterised by an early onset and rapid progression. Both missense mutations in the NLS coding sequence and complete loss of the NLS are known to result in cytoplasmic mislocalisation of FUS protein. However, in addition to the removal of FUS functional domains, frameshift mutations in most cases lead to the attachment of a “tail” of novel amino acids at the FUS C-terminus – a frameshift peptide. It is not clear whether these peptide tails would affect the properties of truncated FUS proteins. In the current study, we compared intracellular behaviour of disease-associated truncated FUS proteins with and without the corresponding frameshift peptides. We demonstrate that some of these peptides can affect subcellular distribution and/or increase aggregation capacity and stability of the truncated FUS protein. Our study suggests that frameshift peptides can alter the properties of truncated FUS variants which may modulate FUS pathogenicity and contribute to the variability of the disease course in ALS-FUS

Defective ryanodine receptor N-terminus inter-subunit interaction is a common mechanism in neuromuscular and cardiac disorders

The ryanodine receptor (RyR) is a homotetrameric channel mediating sarcoplasmic reticulum Ca(2+) release required for skeletal and cardiac muscle contraction. Mutations in RyR1 and RyR2 lead to life-threatening malignant hyperthermia episodes and ventricular tachycardia, respectively. In this brief report, we use chemical cross-linking to demonstrate that pathogenic RyR1 R163C and RyR2 R169Q mutations reduce N-terminus domain (NTD) tetramerization. Introduction of positively-charged residues (Q168R, M399R) in the NTD-NTD inter-subunit interface normalizes RyR2-R169Q NTD tetramerization. These results indicate that perturbation of NTD-NTD inter-subunit interactions is an underlying molecular mechanism in both RyR1 and RyR2 pathophysiology. Importantly, our data provide proof of concept that stabilization of this critical RyR1/2 structure-function parameter offers clear therapeutic potential

Long non-coding RNA Neat1 regulates adaptive behavioural response to stress in mice

NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1−/− mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1−/− mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1−/− mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1−/− neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1−/− mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation

RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca2+ release channel/type 2 ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2R420Q. OBJECTIVE: To determine the arrhythmogenic mechanisms of this mutation. METHODS AND RESULTS: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca2+ waves during confocal Ca2+ recording in both mouse and human RyR2R420Q cardiomyocytes. In addition, Ca2+-induced Ca2+-release, as well as a rough indicator of fractional Ca2+ release, were higher and Ca2+ sparks longer in the RyR2R420Q-expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electronic microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2R420Q binding to junctophilin-2, as there were less junctophilin-2 coimmunoprecipitated with RyR2R420Q. At the single current level, the RyR2R420Q channel dwells longer in the open state at low intracellular Ca2+ ([Ca2+]i), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. CONCLUSIONS: The RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction. GRAPHIC ABSTRACT: An online graphic abstract is available for this article.This work was funded by Inserm and University Paris-Sud, and grants from ANR (ANR-19-CE14-0031-01 to A.M. Gómez), LabEx LERMIT (ANR-10-LABX-33), “Instituto de Salud Carlos III”; FEDER “Union Europea, Una forma de hacer Europa” (PI18/01582), La Fe Biobank (PT17/0015/0043) and Memorial Nacho Barberá to E. Zorio; Swiss National Science Foundation (SNSF grants no. 31003A 179325 and 310030 156375 to E. Niggli), British Heart Foundation (FS/15/30/31494) to S. Zissimopoulos; Italian Telethon ONLUS Foundation (GGP19231) and Italian MIUR (PRIN no. 2015ZZR4W3) to F. Protasi; National Institutes of Health (NIH) grants to J. Ramos-Franco (R01GM111397) and H.H. Valdivia–A.M. Gómez (2R01HL055438-22); and European Union H2020 (MSCA-RISE AMD-734931- 6) to A.M. Gómez. H.H. Valdivia was recipient of a Fullbright-Tockeville chair to work on A.M. Gómez laboratory. J.L. Álvarez and C.R. Valdivia were recipient of a visiting program Alambert from University Paris-Sud to work in A.M. Gómez laboratory. A. Zahradnikova was recipient of a postdoctoral position from University Paris-Sud. R. Rizzetto was recipient of a postdoctoral fellowship from CORDDIM (Région Ile de France). L. Yin was recipient of the CSC (Chinese Scholarship council)