Publications by authors named "Emanuele Buratti"

153 Publications

Aromatic and aliphatic residues of the disordered region of TDP-43 are on a fast track for self-assembly.

Biochem Biophys Res Commun 2021 Nov 20;578:110-114. Epub 2021 Sep 20.

"Rocasolano" Institute for Physical Chemistry, Spanish National Research Council, Serrano 119, 28006, Madrid, Spain. Electronic address:

The C-terminal, intrinsically disordered, prion-like domain (PrLD) of TDP-43 promotes liquid condensate and solid amyloid formation. These phase changes are crucial to the normal biological functions of the protein but also for its abnormal aggregation, which is implicated in amyotrophic lateral sclerosis (ALS) and certain dementias. We and other previously found that certain amyloid forms emerge from an intermediate condensed state that acts as a nucleus for fibrillization. To quantitatively ascertain the role of individual residues within TDP-43's PrLD in its early self-assembly we have followed the kinetics of NMR H-N HSQC signal loss to obtain values for the lag time, elongation rate and extent of condensate formation at equilibrium. The results of this analysis represent a robust corroboration that aliphatic and aromatic residues are key drivers of condensate formation.
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http://dx.doi.org/10.1016/j.bbrc.2021.09.040DOI Listing
November 2021

Diagnostic performance of a colorimetric RT -LAMP for the identification of SARS-CoV-2: A multicenter prospective clinical evaluation in sub-Saharan Africa.

EClinicalMedicine 2021 Oct 28;40:101101. Epub 2021 Aug 28.

International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste 34149, Italy.

Background: Management and control of the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus SARS-CoV-2 is critically dependent on quick and reliable identification of the virus in clinical specimens. Detection of viral RNA by a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a simple, reliable and cost-effective assay, deployable in resource-limited settings (RLS). Our objective was to evaluate the intrinsic and extrinsic performances of RT-LAMP in RLS.

Methods: This is a multicenter prospective observational study of diagnostic accuracy, conducted from October 2020 to February 2021 in four African Countries: Cameroon, Ethiopia, Kenya and Nigeria; and in Italy. We enroled 1657 individuals who were either COVID-19 suspect cases, or asymptomatic and presented for screening. RNA extracted from pharyngeal swabs was tested in parallel by a colorimetric RT-LAMP and by a standard real time polymerase chain reaction (RT-PCR).

Findings: The sensitivity and specificity of index RT LAMP compared to standard RT-PCR on 1657 prospective specimens from infected individuals was determined. For a subset of 1292 specimens, which underwent exactly the same procedures in different countries, we obtained very high specificity (98%) and positive predictive value (PPV = 99%), while the sensitivity was 87%, with a negative predictive value NPV = 70%, Stratification of RT-PCR data showed superior sensitivity achieved with an RT-PCR cycle threshold (Ct) below 35 (97%), which decreased to 60% above 35.

Interpretation: In this field trial, RT-LAMP appears to be a reliable assay, comparable to RT-PCR, particularly with medium-high viral loads (Ct < 35). Hence, RT-LAMP can be deployed in RLS for timely management and prevention of COVID-19, without compromising the quality of output.
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http://dx.doi.org/10.1016/j.eclinm.2021.101101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401528PMC
October 2021

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils.

Viruses 2021 Aug 17;13(8). Epub 2021 Aug 17.

Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), 34136 Trieste, Italy.

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrP, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrP levels to investigate the link between PrP expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrP, PrP, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrP, which encompass almost all pathological amyloids involved in neurodegeneration.
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http://dx.doi.org/10.3390/v13081625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402629PMC
August 2021

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution.

Mol Neurobiol 2021 Aug 14. Epub 2021 Aug 14.

Department of Neurology, Stroke Unit and Laboratory of Neuroscience, Istituto Auxologico Italiano, IRCCS, Piazzale Brescia 20, 20149, Milan, Italy.

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106-110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.
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http://dx.doi.org/10.1007/s12035-021-02505-8DOI Listing
August 2021

HnRNP K mislocalisation is a novel protein pathology of frontotemporal lobar degeneration and ageing and leads to cryptic splicing.

Acta Neuropathol 2021 10 18;142(4):609-627. Epub 2021 Jul 18.

The Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Neuroscience, UCL Queen Square Institute of Neurology, London, UK.

Heterogeneous nuclear ribonucleoproteins (HnRNPs) are a group of ubiquitously expressed RNA-binding proteins implicated in the regulation of all aspects of nucleic acid metabolism. HnRNP K is a member of this highly versatile hnRNP family. Pathological redistribution of hnRNP K to the cytoplasm has been linked to the pathogenesis of several malignancies but, until now, has been underexplored in the context of neurodegenerative disease. Here we show hnRNP K mislocalisation in pyramidal neurons of the frontal cortex to be a novel neuropathological feature that is associated with both frontotemporal lobar degeneration and ageing. HnRNP K mislocalisation is mutually exclusive to TDP-43 and tau pathological inclusions in neurons and was not observed to colocalise with mitochondrial, autophagosomal or stress granule markers. De-repression of cryptic exons in RNA targets following TDP-43 nuclear depletion is an emerging mechanism of potential neurotoxicity in frontotemporal lobar degeneration and the mechanistically overlapping disorder amyotrophic lateral sclerosis. We silenced hnRNP K in neuronal cells to identify the transcriptomic consequences of hnRNP K nuclear depletion. Intriguingly, by performing RNA-seq analysis we find that depletion of hnRNP K induces 101 novel cryptic exon events. We validated cryptic exon inclusion in an SH-SY5Y hnRNP K knockdown and in FTLD brain exhibiting hnRNP K nuclear depletion. We, therefore, present evidence for hnRNP K mislocalisation to be associated with FTLD and for this to induce widespread changes in splicing.
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http://dx.doi.org/10.1007/s00401-021-02340-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423707PMC
October 2021

Clobetasol promotes neuromuscular plasticity in mice after motoneuronal loss via sonic hedgehog signaling, immunomodulation and metabolic rebalancing.

Cell Death Dis 2021 06 16;12(7):625. Epub 2021 Jun 16.

Department of Biomedical and Biotechnological Sciences, Section of Physiology, University of Catania, 95123, Catania, Italy.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.
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http://dx.doi.org/10.1038/s41419-021-03907-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209072PMC
June 2021

Parkin Levels Decrease in Fibroblasts With Progranulin (PGRN) Pathogenic Variants and in a Cellular Model of PGRN Deficiency.

Front Mol Neurosci 2021 13;14:676478. Epub 2021 May 13.

Laboratory of Neurogenetics, Mossakowski Medical Research Institute, Department of Neurodegenerative Disorders, Polish Academy of Sciences, Warsaw, Poland.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with TDP-43 mislocalization and aggregation. Genetic forms of FTLD and ALS are caused by pathogenic variants in various genes, such as (progranulin). To date, depletion of parkin E3 ubiquitin protein ligase, a key mitophagy regulator, has been reported in sporadic ALS patients and ALS mice models with TDP-43 proteinopathy. In this work, we show parkin downregulation also in fibroblasts derived from FTLD patients with four different PGRN pathogenic variants. We corroborate this finding in control fibroblasts upon PGRN silencing, demonstrating additionally the decrease of parkin downstream targets, mitofusin 2 (MFN2) and voltage dependent anion channel 1 (VDAC1). Importantly, we show that TDP-43 overexpression rescues PRKN levels upon transient PGRN silencing, but not in FTLD fibroblasts with PGRN pathogenic variants, despite upregulating PGRN levels in both cases. Further observation of PRKN downregulation upon TDP-43 silencing, suggests that TDP-43 loss-of-function contributes to PRKN decrease. Our results provide further evidence that parkin downregulation might be a common and systemic phenomenon in neurodegenerative diseases with TDP- 43 loss-of-function.
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http://dx.doi.org/10.3389/fnmol.2021.676478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155584PMC
May 2021

State-of-the-Art Methods and Emerging Fluid Biomarkers in the Diagnostics of Dementia-A Short Review and Diagnostic Algorithm.

Diagnostics (Basel) 2021 Apr 27;11(5). Epub 2021 Apr 27.

Neurology Unit, Department of Clinical and Experimental Sciences, University of Brescia, 25121 Brescia, Italy.

The most common neurodegenerative dementias include Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). The correct etiology-based diagnosis is pivotal for clinical management of these diseases as well as for the suitable timing and choosing the accurate disease-modifying therapies when these become available. Enzyme-linked immunosorbent assay (ELISA)-based methods, detecting altered levels of cerebrospinal fluid (CSF) Tau, phosphorylated Tau, and Aβ-42 in AD, allowed the wide use of this set of biomarkers in clinical practice. These analyses demonstrate a high diagnostic accuracy in AD but suffer from a relatively restricted usefulness due to invasiveness and lack of prognostic value. In recent years, the development of novel advanced techniques has offered new state-of-the-art opportunities in biomarker discovery. These include single molecule array technology (SIMOA), a tool for non-invasive analysis of ultra-low levels of central nervous system-derived molecules from biofluids, such as CSF or blood, and real-time quaking (RT-QuIC), developed to analyze misfolded proteins. In the present review, we describe the history of methods used in the fluid biomarker analyses of dementia, discuss specific emerging biomarkers with translational potential for clinical use, and suggest an algorithm for the use of new non-invasive blood biomarkers in clinical practice.
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http://dx.doi.org/10.3390/diagnostics11050788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145467PMC
April 2021

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

PLoS Biol 2021 04 28;19(4):e3001198. Epub 2021 Apr 28.

Rocasolano Institute of Physical Chemistry, Spanish National Research Council, Madrid, Spain.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.
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http://dx.doi.org/10.1371/journal.pbio.3001198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109789PMC
April 2021

Cerebrospinal fluid from frontotemporal dementia patients is toxic to neurons.

Biochim Biophys Acta Mol Basis Dis 2021 06 11;1867(6):166122. Epub 2021 Mar 11.

Department of Experimental Neuroscience, IRCCS Fondazione Santa Lucia, Rome, Italy. Electronic address:

Frontotemporal Lobar Degeneration (FTD) is a neurodegenerative disease characterized by a progressive deterioration of cognitive functions. Currently, no effective treatment exists. We have studied cytotoxicity and neuronal functionality in cortical and spinal cord cultures upon exposure to cerebrospinal fluid (CSF) from 39 FTD patients. FTD-CSF alters the miniature excitatory postsynaptic currents in the cortical cultures and it is toxic to spinal cord cultures, particularly to GABAergic+ and calbindin-D28k + neurons.
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http://dx.doi.org/10.1016/j.bbadis.2021.166122DOI Listing
June 2021

Trends in Understanding the Pathological Roles of TDP-43 and FUS Proteins.

Authors:
Emanuele Buratti

Adv Exp Med Biol 2021 ;1281:243-267

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.

Following the discovery of TDP-43 and FUS involvement in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), the major challenge in the field has been to understand their physiological functions, both in normal and disease conditions. The hope is that this knowledge will improve our understanding of disease and lead to the development of effective therapeutic options. Initially, the focus has been directed at characterizing the role of these proteins in the control of RNA metabolism, because the main function of TDP-43 and FUS is to bind coding and noncoding RNAs to regulate their life cycle within cells. As a result, we now have an in-depth picture of the alterations that occur in RNA metabolism following their aggregation in various ALS/FTLD models and, to a somewhat lesser extent, in patients' brains. In parallel, progress has been made with regard to understanding how aggregation of these proteins occurs in neurons, how it can spread in different brain regions, and how these changes affect various metabolic cellular pathways to result in neuronal death. The aim of this chapter will be to provide a general overview of the trending topics in TDP-43 and FUS investigations and to highlight what might represent the most promising avenues of research in the years to come.
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http://dx.doi.org/10.1007/978-3-030-51140-1_15DOI Listing
February 2021

Deferoxamine mesylate improves splicing and GAA activity of the common c.-32-13T>G allele in late-onset PD patient fibroblasts.

Mol Ther Methods Clin Dev 2021 Mar 20;20:227-236. Epub 2020 Nov 20.

Centre for Rare Diseases, Academic Hospital Santa Maria della Misericordia, Udine, Italy.

Pompe disease (PD) is an autosomal recessive lysosomal storage disorder due to deficient activity of the acid alpha glucosidase enzyme (GAA). As a consequence of the enzymatic defect, undigested glycogen accumulates within lysosomes. Most patients affected by the late-onset (LO) phenotype carry in at least one allele the c.-32-13T>G variant, which leads to exon 2 exclusion from the pre-mRNA. These patients display a variable and suboptimal response to enzyme replacement therapy. To identify novel therapeutic approaches, we developed a fluorescent GAA exon 2 splicing assay and screened a library of US Food and Drug Administration (FDA)-approved compounds. This led to the identification of several drugs able to restore normal splicing. Among these, we further validated the effects of the iron chelator deferoxamine (Defe) in c.-32-13T>G fibroblasts. Defe treatment resulted in a 2-fold increase of GAA exon 2 inclusion and a 40% increase in enzymatic activity. Preliminary results suggest that this effect is mediated by the regulation of iron availability, at least partially. RNA-seq experiments also showed that Defe might shift the balance of splicing factor levels toward a profile promoting GAA exon 2 inclusion. This work provides the basis for drug repurposing and development of new chemically modified molecules aimed at improving the clinical outcome in LO-PD patients.
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http://dx.doi.org/10.1016/j.omtm.2020.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782201PMC
March 2021

NMR assignments for the C-terminal domain of human TDP-43.

Biomol NMR Assign 2021 04 8;15(1):177-181. Epub 2021 Jan 8.

"Rocasolano" Institute for Physical Chemistry, Spanish National Research Council, Serrano 119, 28006, Madrid, Spain.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a 414-residue protein whose aberrant aggregation is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Intriguingly, TDP-43 has also been shown to functionally oligomerize to carry out physiological functions. TDP-43 also exists in mixed condensates or granules with other proteins (e.g. neuronal or stress granules), and its large C-terminal domain (CTD, residues 267-414) seems responsible for TDP-43 both homo- and heterotypic interactions underlying such diverse functional and pathological aggregation events. A myriad of distinct triggers may drive TDP-43 oligomerization, including interaction partners or changes in pH or salinity. In this Assignment Note, we report the complete backbone and a wealth of side chain chemical shift assignments for the CTD of TDP-43 at pH 4. The assignments presented here provide a solid starting point to study the aggregation pathway of TDP-43 at pH values below those considered physiological but relevant in pathological settings, and to contrast the aggregation behaviour under distinct conditions and in the presence of interacting partners.
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http://dx.doi.org/10.1007/s12104-020-10002-7DOI Listing
April 2021

Editorial: RNA Splicing and Backsplicing: Disease and Therapy.

Front Genet 2020 8;11:626835. Epub 2020 Dec 8.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.

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http://dx.doi.org/10.3389/fgene.2020.626835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753067PMC
December 2020

Somatic TARDBP variants as a cause of semantic dementia.

Brain 2020 12;143(12):3827-3841

Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands.

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants in neurodegenerative disease. Semantic dementia is characterized by a non-familial occurrence, early onset (<65 years), focal temporal atrophy and TDP-43 pathology. To test whether somatic variants in neural progenitor cells during brain development might lead to semantic dementia, we compared deep exome sequencing data of DNA derived from brain and blood of 16 semantic dementia cases. Somatic variants observed in brain tissue and absent in blood were validated using amplicon sequencing and digital PCR. We identified two variants in exon one of the TARDBP gene (L41F and R42H) at low level (1-3%) in cortical regions and in dentate gyrus in two semantic dementia brains, respectively. The pathogenicity of both variants is supported by demonstrating impaired splicing regulation of TDP-43 and by altered subcellular localization of the mutant TDP-43 protein. These findings indicate that somatic variants may cause semantic dementia as a non-hereditary neurodegenerative disease, which might be exemplary for other late-onset neurodegenerative disorders.
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http://dx.doi.org/10.1093/brain/awaa317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805802PMC
December 2020

TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

Brain Commun 2020 14;2(2):fcaa142. Epub 2020 Sep 14.

Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Laboratory of Prion Biology, Trieste, Italy.

The pathological deposition of the transactive response DNA-binding protein of 43 kDa occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments capable of interfering with the underlying pathological process and early detection of transactive response DNA-binding protein of 43 kDa pathology would facilitate the development of disease-modifying drugs. The real-time quaking-induced conversion reaction showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite transactive response DNA-binding protein of 43 kDa displays prion-like properties, to date the real-time quaking-induced conversion reaction technology has not yet been adapted to this protein. The aim of this study was to adapt the real-time quaking-induced conversion reaction technique for the transactive response DNA-binding protein of 43 kDa substrate and to exploit the intrinsic ability of this technology to amplify minute amount of mis-folded proteins for the detection of pathological transactive response DNA-binding protein of 43 kDa species in the cerebrospinal fluid of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic transactive response DNA-binding protein of 43 kDa-pre-formed aggregates and with autopsy-verified brain homogenate samples and subsequently analysed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction was able to detect as little as 15 pg of transactive response DNA-binding protein of 43 kDa aggregates, discriminating between a cohort of patients affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that transactive response DNA-binding protein of 43 kDa is a suitable substrate for the real-time quaking-induced conversion reaction. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The cerebrospinal fluid detection of transactive response DNA-binding protein of 43 kDa pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.
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http://dx.doi.org/10.1093/braincomms/fcaa142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566418PMC
September 2020

Antibody against TDP-43 phosphorylated at serine 375 suggests conformational differences of TDP-43 aggregates among FTLD-TDP subtypes.

Acta Neuropathol 2020 11 10;140(5):645-658. Epub 2020 Aug 10.

Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada.

Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD-TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS-TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD-TDP and ALS-TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD-TDP and ALS-TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD-TDP subtypes examined (A-C) and in ALS-TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43 antiserum among pathological subtypes. TDP-43 pathology of ALS-TDP, FTLD-TDP type B (including cases with the C9orf72 mutation), and FTLD-TDP type C all showed strong pTDP-43 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD-TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers (i.e., TDP-43 strains) as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.
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http://dx.doi.org/10.1007/s00401-020-02207-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547034PMC
November 2020

From basic research to the clinic: innovative therapies for ALS and FTD in the pipeline.

Mol Neurodegener 2020 06 1;15(1):31. Epub 2020 Jun 1.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149, Trieste, Italy.

Amyotrophic lateral sclerosis (ALS) and Frontotemporal Degeneration (FTD) are neurodegenerative disorders, related by deterioration of motor and cognitive functions and short survival. Aside from cases with an inherited pathogenic mutation, the causes of the disorders are still largely unknown and no effective treatment currently exists. It has been shown that FTD may coexist with ALS and this overlap occurs at clinical, genetic, and molecular levels. In this work, we review the main pathological aspects of these complex diseases and discuss how the integration of the novel pathogenic molecular insights and the analysis of molecular interaction networks among all the genetic players represents a critical step to shed light on discovering novel therapeutic strategies and possibly tailoring personalized medicine approaches to specific ALS and FTD patients.
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http://dx.doi.org/10.1186/s13024-020-00373-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268618PMC
June 2020

Targeting TDP-43 proteinopathy with drugs and drug-like small molecules.

Authors:
Emanuele Buratti

Br J Pharmacol 2021 03 30;178(6):1298-1315. Epub 2020 Jun 30.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.

Following the discovery of the involvement of the ribonucleoprotein TDP-43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a major research focus has been to develop treatments that can prevent or alleviate these disease conditions. One pharmacological approach has been to use TDP-43-based disease models to test small molecules and drugs already known to have some therapeutic effect in a variety of neurodegenerative conditions. In parallel, various disease models have been used to perform high-throughput screens of drugs and small compound libraries. The aim of this review will be to provide a general overview of the compounds that have been described to alter pathological characteristics of TDP-43. These include expression levels, cytoplasmic mis-localization, post-translational modifications, cleavage, stress granule recruitment and aggregation. In parallel, this review will also address the use of compounds that modify the autophagic/proteasome systems that are known to target TDP-43 misfolding and aggregation. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.
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http://dx.doi.org/10.1111/bph.15148DOI Listing
March 2021

Pre-mRNA splicing defects and RNA binding protein involvement in Niemann Pick type C disease.

J Biotechnol 2020 Jul 6;318:20-30. Epub 2020 May 6.

Molecular Pathology, International Institute for Genetic Engineering and Biotechnology, Trieste, Italy. Electronic address:

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disorder due to mutations in NPC1 (95 % cases) or NPC2 genes, encoding NPC1 and NPC2 proteins, respectively. Both NPC1 and NPC2 proteins are involved in transport of intracellular cholesterol and their alteration leads to the accumulation of unesterified cholesterol and other lipids within the lysosomes. The disease is characterized by visceral, neurological and psychiatric symptoms. However, the pathogenic mechanisms that lead to the fatal neurodegeneration are still unclear. To date, several mutations leading to the generation of aberrant splicing variants or mRNA degradation in NPC1 and NPC2 genes have been reported. In addition, different lines of experimental evidence have highlighted the possible role of RNA-binding proteins and RNA-metabolism, in the onset and progression of many neurodegenerative disorders, that could explain NPC neurological features and in general, the disease pathogenesis. In this review, we will provide an overview of the impact of mRNA processing and metabolism on NPC disease pathology.
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http://dx.doi.org/10.1016/j.jbiotec.2020.03.012DOI Listing
July 2020

Neurodegeneration: The emerging non-coding connections.

Noncoding RNA Res 2019 Sep 1;4(3):79. Epub 2019 Oct 1.

Molecular Pathology, International Institute for Genetic Engineering and Biotechnology, Trieste, Italy.

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http://dx.doi.org/10.1016/j.ncrna.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926175PMC
September 2019

Editorial: Role of RNA Modification in Disease.

Front Genet 2019 23;10:920. Epub 2019 Oct 23.

Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Pathology, New York University School of Medicine, New York, NY, United States.

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http://dx.doi.org/10.3389/fgene.2019.00920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819427PMC
October 2019

Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology.

Hum Mutat 2019 11 29;40(11):2121-2130. Epub 2019 Jul 29.

Molecular Pathology, International Institute for Genetic Engineering and Biotechnology, Trieste, Italy.

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.
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http://dx.doi.org/10.1002/humu.23867DOI Listing
November 2019

Toward a Glutamate Hypothesis of Frontotemporal Dementia.

Front Neurosci 2019 29;13:304. Epub 2019 Mar 29.

Neurology Unit, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy.

Frontotemporal dementia (FTD) is a heterogenous neurodegenerative disorder, characterized by diverse clinical presentations, neuropathological characteristics and underlying genetic causes. Emerging evidence has shown that FTD is characterized by a series of changes in several neurotransmitter systems, including serotonin, dopamine, GABA and, above all, glutamate. Indeed, several studies have now provided preclinical and clinical evidence that glutamate is key in the pathogenesis of FTD. Animal models of FTD have shown a selective hypofunction in -methyl -aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, while in patients, glutamatergic pyramidal neurons are depleted in several areas, including the frontal and temporal cortices. Recently, a selective involvement of the AMPA GluA3 subunit has been observed in patients with autoimmune anti-GluA3 antibodies, which accounted for nearly 25% of FTD patients, leading to a decrease of the GluA3 subunit synaptic localization of the AMPA receptor and loss of dendritic spines. Other evidence of the involvement of the glutamatergic system in FTD derives from non-invasive brain stimulation studies using transcranial magnetic stimulation, in which specific stimulation protocols have indirectly identified a selective and prominent impairment in glutamatergic circuits in patients with both sporadic and genetic FTD. In view of limited disease modifying therapies to slow or revert disease progression in FTD, an important approach could consist in targeting the neurotransmitter deficits, similarly to what has been achieved in Parkinson's disease with dopaminergic therapy or Alzheimer's disease with cholinergic therapy. In this review, we summarize the current evidence concerning the involvement of the glutamatergic system in FTD, suggesting the development of new therapeutic strategies.
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http://dx.doi.org/10.3389/fnins.2019.00304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449454PMC
March 2019

Mis-splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays.

J Pathol 2019 08 23;248(4):409-420. Epub 2019 Apr 23.

Splicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, Spain.

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5268DOI Listing
August 2019

Dysregulation of TDP-43 intracellular localization and early onset ALS are associated with a TARDBP S375G variant.

Brain Pathol 2019 05 27;29(3):397-413. Epub 2018 Dec 27.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.

We investigated the Central Nervous System (CNS) and skeletal muscle tissue from A woman was clinically diagnosed with amyotrophic lateral sclerosis (ALS) at the age of 22. Neuropathologic evaluation showed upper and lower motor neuron loss, corticospinal tract degeneration and skeletal muscle denervation. Analysis of the patient's Deoxyribonucleic acid (DNA) revealed a AGT>GGT change resulting in an S375G substitution in the C-terminal region of TDP-43. This variant was previously reported as being benign. Considering the early onset and severity of the disease in this patient, we tested the effects of this genetic variant on TDP-43 localization, pre-mRNA splicing activity and toxicity, in parallel with the effects on known neighboring disease-associated mutations. In cell lines, expressed in culture, S375G TDP-43 appeared to be more significantly localized in the nucleus and to exert higher toxicity than wild-type TDP-43. Strikingly, a phosphomimic mutant at the same residue (S375E) showed a strong tendency to accumulate in the cytoplasm, especially under stress conditions, and molecular dynamics simulations suggest that phosphorylation of this residue can disrupt TDP-43 intermolecular interactions. The results of the current study highlight the importance of phosphorylation and regulation of TDP-43 nuclear-cytoplasmic shuttling/redistribution, in relation to the pathogenetic mechanisms involved in different forms of ALS.
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http://dx.doi.org/10.1111/bpa.12680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6875182PMC
May 2019

Systematic Analysis of Gene Expression Profiles Controlled by hnRNP Q and hnRNP R, Two Closely Related Human RNA Binding Proteins Implicated in mRNA Processing Mechanisms.

Front Mol Biosci 2018 30;5:79. Epub 2018 Aug 30.

Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

Heteregeneous ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that take part in all processes that involve mRNA maturation. As a consequence, alterations of their homeostasis may lead to many complex pathological disorders, such as neurodegeneration and cancer. For many of these proteins, however, their exact function and cellular targets are still not very well known. Here, we focused the attention on two hnRNP family members, hnRNP Q and hnRNP R, that we previously found affecting TDP-43 activity both in Drosophila melanogaster and human neuronal cell line. Classification of these two human proteins as paralogs is suported by the high level of sequence homology and by the observation that in fly they correspond to the same protein, namely Syp. We profiled differentially expressed genes from RNA-Seq and generated functional enrichment results after silencing of hnRNP Q and hnRNP R in neuroblastoma SH-SY5Y cell line. Interestingly, despite their high sequence similarity, these two proteins were found to affect different cellular pathways, especially with regards to neurodegeneration, such as PENK, NGR3, RAB26, JAG1, as well as inflammatory response, such as TNF, ICAM1, ICAM5, and TNFRSF9. In conclusion, human hnRNP Q and hnRNP R may be considered potentially important regulators of neuronal homeostasis and their disruption could impair distinct pathways in the central nervous system axis, thus confirming the importance of their conservation during evolution.
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http://dx.doi.org/10.3389/fmolb.2018.00079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125337PMC
August 2018

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

EMBO J 2018 06 15;37(11). Epub 2018 May 15.

UCL Institute of Neurology and Francis Crick Institute, London, UK.

TDP-43 (encoded by the gene ) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous to dissect TDP-43 function at physiological levels both and Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. , this gain-of-function mutation in endogenous causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.
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http://dx.doi.org/10.15252/embj.201798684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983119PMC
June 2018

TDP-43 post-translational modifications in health and disease.

Authors:
Emanuele Buratti

Expert Opin Ther Targets 2018 03 20;22(3):279-293. Epub 2018 Feb 20.

a Department of Molecular Pathology , International Centre for Genetic Engineering and Biotechnology (ICGEB) , Trieste , Italy.

Introduction: Nuclear factor TDP-43 is a ubiquitously expressed RNA binding protein that plays a key causative role in several neurodegenerative diseases, especially in the ALS/FTD spectrum. In addition, its aberrant aggregation and expression has been recently observed in other type of diseases, such as myopathies and Niemann-Pick C, a lysosomal storage disease. Areas covered: This review aims to specifically cover the post-translational modifications (PTMs) that can affect TDP-43 function and cellular status both in health and disease. To this date, these include phosphorylation, formation of C-terminal fragments, disulfide bridge formation, ubiquitination, acetylation, and sumoylation. Recently published articles on these subjects have been reviewed in this manuscript. Expert opinion: Targeting aberrant TDP-43 expression in neurodegenerative diseases is a very challenging task due to the fact that both its overexpression and downregulation are considerably toxic to cells. This characteristic makes it difficult to therapeutically target this protein in a generalized manner. An alternative approach could be the identification of specific aberrant PTMs that promote its aggregation or toxicity, and developing novel therapeutic approaches toward their selective modification.
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http://dx.doi.org/10.1080/14728222.2018.1439923DOI Listing
March 2018
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