Title: Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice
Abstract: Article8 June 2020Open Access Source DataTransparent process Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice Carlo Wilke Carlo Wilke orcid.org/0000-0002-7250-8597 Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Eva Haas Eva Haas Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany Centre for Rare Diseases, University of Tübingen, Tübingen, Germany Search for more papers by this author Kathrin Reetz Kathrin Reetz Department of Neurology, RWTH Aachen University, Aachen, Germany JARA-BRAIN Institute Molecular Neuroscience and Neuroimaging, Forschungszentrum Jülich, RWTH Aachen University, Aachen, Germany Search for more papers by this author Jennifer Faber Jennifer Faber Department of Neurology, University Hospital Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Hector Garcia-Moreno Hector Garcia-Moreno Ataxia Centre, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Magda M Santana Magda M Santana Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Bart van de Warrenburg Bart van de Warrenburg Donders Institute for Brain, Cognition, and Behaviour, Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Holger Hengel Holger Hengel Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Manuela Lima Manuela Lima Faculdade de Ciências e Tecnologia, Universidade dos Açores, Ponta Delgada, Portugal Search for more papers by this author Alessandro Filla Alessandro Filla Department of Neuroscience, and Reproductive and Odontostomatological Sciences, Federico II University Naples, Naples, Italy Search for more papers by this author Alexandra Durr Alexandra Durr Sorbonne Université, Institut du Cerveau et de la Moelle épinière (ICM), AP-HP, Inserm, CNRS, University Hospital Pitié-Salpêtrière, Paris, France Search for more papers by this author Bela Melegh Bela Melegh Department of Medical Genetics, Szentagothai Research Center, University of Pécs Medical School, Pécs, Hungary Search for more papers by this author Marcella Masciullo Marcella Masciullo Spinal Rehabilitation Lab (SPIRE), IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Jon Infante Jon Infante Service of Neurology, University Hospital Marqués de Valdecilla (IDIVAL), University of Cantabria (UC), Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Santander, Spain Search for more papers by this author Paola Giunti Paola Giunti Ataxia Centre, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Manuela Neumann Manuela Neumann German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Department of Neuropathology, University of Tübingen, Tübingen, Germany Search for more papers by this author Jeroen de Vries Jeroen de Vries Department of Neurology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Luis Pereira de Almeida Luis Pereira de Almeida orcid.org/0000-0001-5831-3307 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Maria Rakowicz Maria Rakowicz First Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland Search for more papers by this author Heike Jacobi Heike Jacobi German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurology, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Rebecca Schüle Rebecca Schüle Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Stephan A Kaeser Stephan A Kaeser Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Jens Kuhle Jens Kuhle Neurology, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Thomas Klockgether Thomas Klockgether Department of Neurology, University Hospital Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Ludger Schöls Ludger Schöls Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author SCA3 neurofilament study group SCA3 neurofilament study groupSee Appendix 1Search for more papers by this author Christian Barro Christian Barro Neurology, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Jeannette Hübener-Schmid Jeannette Hübener-Schmid Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany Centre for Rare Diseases, University of Tübingen, Tübingen, Germany Search for more papers by this author Matthis Synofzik Corresponding Author Matthis Synofzik [email protected] orcid.org/0000-0002-2280-7273 Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Carlo Wilke Carlo Wilke orcid.org/0000-0002-7250-8597 Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Eva Haas Eva Haas Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany Centre for Rare Diseases, University of Tübingen, Tübingen, Germany Search for more papers by this author Kathrin Reetz Kathrin Reetz Department of Neurology, RWTH Aachen University, Aachen, Germany JARA-BRAIN Institute Molecular Neuroscience and Neuroimaging, Forschungszentrum Jülich, RWTH Aachen University, Aachen, Germany Search for more papers by this author Jennifer Faber Jennifer Faber Department of Neurology, University Hospital Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Hector Garcia-Moreno Hector Garcia-Moreno Ataxia Centre, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Magda M Santana Magda M Santana Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Bart van de Warrenburg Bart van de Warrenburg Donders Institute for Brain, Cognition, and Behaviour, Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Holger Hengel Holger Hengel Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Manuela Lima Manuela Lima Faculdade de Ciências e Tecnologia, Universidade dos Açores, Ponta Delgada, Portugal Search for more papers by this author Alessandro Filla Alessandro Filla Department of Neuroscience, and Reproductive and Odontostomatological Sciences, Federico II University Naples, Naples, Italy Search for more papers by this author Alexandra Durr Alexandra Durr Sorbonne Université, Institut du Cerveau et de la Moelle épinière (ICM), AP-HP, Inserm, CNRS, University Hospital Pitié-Salpêtrière, Paris, France Search for more papers by this author Bela Melegh Bela Melegh Department of Medical Genetics, Szentagothai Research Center, University of Pécs Medical School, Pécs, Hungary Search for more papers by this author Marcella Masciullo Marcella Masciullo Spinal Rehabilitation Lab (SPIRE), IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Jon Infante Jon Infante Service of Neurology, University Hospital Marqués de Valdecilla (IDIVAL), University of Cantabria (UC), Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Santander, Spain Search for more papers by this author Paola Giunti Paola Giunti Ataxia Centre, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Manuela Neumann Manuela Neumann German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Department of Neuropathology, University of Tübingen, Tübingen, Germany Search for more papers by this author Jeroen de Vries Jeroen de Vries Department of Neurology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Luis Pereira de Almeida Luis Pereira de Almeida orcid.org/0000-0001-5831-3307 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Maria Rakowicz Maria Rakowicz First Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland Search for more papers by this author Heike Jacobi Heike Jacobi German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurology, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Rebecca Schüle Rebecca Schüle Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Stephan A Kaeser Stephan A Kaeser Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Jens Kuhle Jens Kuhle Neurology, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Thomas Klockgether Thomas Klockgether Department of Neurology, University Hospital Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Ludger Schöls Ludger Schöls Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author SCA3 neurofilament study group SCA3 neurofilament study groupSee Appendix 1Search for more papers by this author Christian Barro Christian Barro Neurology, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Jeannette Hübener-Schmid Jeannette Hübener-Schmid Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany Centre for Rare Diseases, University of Tübingen, Tübingen, Germany Search for more papers by this author Matthis Synofzik Corresponding Author Matthis Synofzik [email protected] orcid.org/0000-0002-2280-7273 Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany Search for more papers by this author Author Information Carlo Wilke1,2, Eva Haas3,4, Kathrin Reetz5,6, Jennifer Faber7,8, Hector Garcia-Moreno9,10, Magda M Santana11, Bart Warrenburg12, Holger Hengel1,2, Manuela Lima13, Alessandro Filla14, Alexandra Durr15, Bela Melegh16, Marcella Masciullo17, Jon Infante18, Paola Giunti9,10, Manuela Neumann2,19, Jeroen Vries20, Luis Pereira de Almeida11, Maria Rakowicz21, Heike Jacobi8,22, Rebecca Schüle1,2, Stephan A Kaeser1,2, Jens Kuhle23, Thomas Klockgether7,8, Ludger Schöls1,2, , Christian Barro23,‡, Jeannette Hübener-Schmid3,4,‡, Matthis Synofzik *,1,2,‡, Christian Deuschle, Elke Stransky, Kathrin Brockmann, Jörg B Schulz, Laszlo Baliko, Judith Gaalen, Mafalda Raposo and Andreas Jeromin 1Hertie Institute for Clinical Brain Research (HIH), Center of Neurology, University of Tübingen, Tübingen, Germany 2German Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany 3Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany 4Centre for Rare Diseases, University of Tübingen, Tübingen, Germany 5Department of Neurology, RWTH Aachen University, Aachen, Germany 6JARA-BRAIN Institute Molecular Neuroscience and Neuroimaging, Forschungszentrum Jülich, RWTH Aachen University, Aachen, Germany 7Department of Neurology, University Hospital Bonn, Bonn, Germany 8German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 9Ataxia Centre, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK 10National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK 11Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 12Donders Institute for Brain, Cognition, and Behaviour, Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands 13Faculdade de Ciências e Tecnologia, Universidade dos Açores, Ponta Delgada, Portugal 14Department of Neuroscience, and Reproductive and Odontostomatological Sciences, Federico II University Naples, Naples, Italy 15Sorbonne Université, Institut du Cerveau et de la Moelle épinière (ICM), AP-HP, Inserm, CNRS, University Hospital Pitié-Salpêtrière, Paris, France 16Department of Medical Genetics, Szentagothai Research Center, University of Pécs Medical School, Pécs, Hungary 17Spinal Rehabilitation Lab (SPIRE), IRCCS Fondazione Santa Lucia, Rome, Italy 18Service of Neurology, University Hospital Marqués de Valdecilla (IDIVAL), University of Cantabria (UC), Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Santander, Spain 19Department of Neuropathology, University of Tübingen, Tübingen, Germany 20Department of Neurology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands 21First Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland 22Department of Neurology, University Hospital of Heidelberg, Heidelberg, Germany 23Neurology, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland ‡These authors contributed equally to this work as last authors *Corresponding author. Tel: +49 7071 2982060; E-mail: [email protected] EMBO Mol Med (2020)12:e11803https://doi.org/10.15252/emmm.201911803 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract With molecular treatments coming into reach for spinocerebellar ataxia type 3 (SCA3), easily accessible, cross-species validated biomarkers for human and preclinical trials are warranted, particularly for the preataxic disease stage. We assessed serum levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in ataxic and preataxic subjects of two independent multicentric SCA3 cohorts and in a SCA3 knock-in mouse model. Ataxic SCA3 subjects showed increased levels of both NfL and pNfH. In preataxic subjects, NfL levels increased with proximity to the individual expected onset of ataxia, with significant NfL elevations already 7.5 years before onset. Cross-sectional NfL levels correlated with both disease severity and longitudinal disease progression. Blood NfL and pNfH increases in human SCA3 were each paralleled by similar changes in SCA3 knock-in mice, here also starting already at the presymptomatic stage, closely following ataxin-3 aggregation and preceding Purkinje cell loss in the brain. Blood neurofilaments, particularly NfL, might thus provide easily accessible, cross-species validated biomarkers in both ataxic and preataxic SCA3, associated with earliest neuropathological changes, and serve as progression, proximity-to-onset and, potentially, treatment-response markers in both human and preclinical SCA3 trials. Synopsis This cross-species study establishes neurofilament blood levels (NfL/pNfH) as biomarkers of neuronal damage in spinocerebellar ataxia type 3 (SCA3) in humans and mice, both at the manifest and premanifest disease stage. NfL levels capture proximity to symptom onset and disease progression. Blood levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) were assessed in manifest and premanifest subjects of two multicentric SCA3 cohorts and in SCA3 knock-in mice. NfL and pNfH levels were increased at the manifest disease stage, with NfL levels reflecting both clinical disease severity and disease progression. NfL elevations in the premanifest stage were present already 7.5 years before the individual expected onset, with levels increasing further in temporal proximity to symptom onset. NfL and pNfH increases in the SCA3 mouse model started also already at the premanifest stage, closely following ataxin-3 aggregation and even preceding Purkinje cell loss in the brain. The paper explained Problem Spinocerebellar ataxia type 3 (SCA3), the most frequent autosomal-dominant ataxia worldwide, is a prototypic neurodegenerative repeat-expansion disorder. As targeted molecular treatments for SCA3 (e.g. antisense oligonucleotides) are coming into reach, easily accessible peripheral biomarkers are warranted, both for human and for preclinical trials. Such biomarkers are particularly important at the presymptomatic disease stage, where disease-modifying therapies might be most effective, and require cross-validation in animal models, as well as cross-validation with associated central nervous system changes. Results In two independent multicentric human SCA3 cohorts, blood levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) were each increased at the symptomatic disease stage. NfL levels were increased also at the presymptomatic stage. NfL elevations were present already 7.5 years before the individual expected symptom onset, with levels increasing further in proximity to the conversion from the presymptomatic to the symptomatic disease stage. NfL levels reflected both subjects’ clinical disease severity and disease progression. The neurofilament increases in our human cohorts were paralleled by neurofilament increases in a SCA3 knock-in mouse model, here also starting already at the presymptomatic stage, closely following the onset of ataxin-3 aggregation and preceding significant Purkinje cell loss in the brain. These results allowed mapping a larger biomarker cascade of SCA3 disease, capturing differential changes in NfL, pNfH, ataxin-3 and behavioural biomarker trajectories across disease stages. Impact Blood levels of neurofilaments, particularly NfL, might provide easily accessible peripheral biomarkers of neuronal damage in SCA3, validated both at the presymptomatic and at symptomatic disease stage in humans and mice and associated with brain pathology changes already at the earliest disease stages. NfL levels might serve as progression, proximity-to-onset and, potentially, treatment-response biomarkers for both human and preclinical trials. Introduction Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, is the most common dominantly inherited form of degenerative ataxia, caused by an expanded CAG repeat in the ATXN3 gene and marked by irreversible decline in motor function already in mid-life (Costa Mdo & Paulson, 2012; Rub et al, 2013). Advances in the understanding of the toxic gain-of-function mechanisms underlying SCA3 neurodegeneration have opened a window for targeted molecular therapies (Paulson et al, 2017; Ramani et al, 2017). Particularly, interventions with antisense oligonucleotides (ASOs) targeting mutated ATXN3 show promising results in mitigating the molecular, pathological and behavioural disease-associated changes in a SCA3 mouse model (McLoughlin et al, 2018). ASO treatments might allow preventing the neurodegenerative process even before the occurrence of clinical symptoms (Finkel et al, 2017; Winter et al, 2019). However, to pave the way for upcoming trials of these promising therapies, easily accessible, objective and sensitive outcome parameters are urgently needed to track disease progression in both the preataxic and ataxic stage of SCA3 disease. Such parameters require validation in large human SCA3 cohorts with standardised phenotyping and in SCA3 mouse models, as mouse models allow comprehensive neuropathological validation and preclinical treatment trials, even already during the presymptomatic stage. In this cross-species biomarker study, we propose serum concentrations of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) as easily accessible, objective and sensitive blood biomarkers of disease severity in SCA3. Neurofilaments (Nfs) are neuron-specific cytoskeletal proteins, with constant low-amount release from neurons in an age-related manner (Disanto et al, 2017; Gaetani et al, 2019; Khalil et al, 2020) and rapid increase in response to axonal damage irrespective of the underlying cause (e.g. traumatic, vascular, inflammatory, degenerative injury). It is yet unknown whether Nf elevations relate to passive release from damaged axons and/or upregulated protein production and secretion reflecting attempted axonal regeneration (Paterson et al, 2019). With novel ultra-sensitive single-molecule array (Simoa) assays, Nfs are now reliably quantifiable in peripheral blood (Wilke et al, 2016; Khalil et al, 2018). In fact, while the correlation between Nf levels in the extracellular compartment of the brain and the cerebrospinal fluid (CSF) is yet unknown, a close correlation between Nf levels in the CSF and the peripheral blood has been established for several neurodegenerative diseases (Bacioglu et al, 2016; Wilke et al, 2016, 2019; Khalil et al, 2018; Gaetani et al, 2019), including also SCA3 (Li et al, 2019). Hereby, Nf levels in blood are roughly 2.5% of the levels in the CSF (Disanto et al, 2017). Our previous work in a mixed cohort of repeat-expansion spinocerebellar ataxias (SCAs) indicated that blood concentrations of NfL in multisystemic repeat SCAs are increased at the ataxic disease stage (Wilke et al, 2018), and this also been reported specifically for SCA3 (Li et al, 2019). However, all these previous studies were confined to single-centre, single-assay and human-only assessments without any neuropathology associations, as well as to cross-sectional data. Moreover, they were all restricted to NfL only, while pNfH might also allow capturing neuronal disintegration and particularly axonal damage in neurodegenerative disease, possibly capturing differential features of the neurodegenerative process compared to NfL (Khalil et al, 2018; Wilke et al, 2019). We here hypothesised that both serum Nfs might serve as blood biomarkers of disease severity in both human SCA3 and mouse models, expecting increased concentrations at both the ataxic and preataxic stage, with increases in preataxic subjects occurring particularly in proximity to the onset of ataxia. We measured serum concentrations of both Nfs in cross-sectional samples of ataxic and preataxic SCA3 subjects and controls in two independent multicentric cohorts, using two independent ultra-sensitive single-molecule array (Simoa) approaches for each of both Nfs, and correlated Nf levels with measures of disease severity. We expected the blood Nf increases in human SCA3 to be paralleled by blood Nf increases in SCA3 animal models, also starting already in the presymptomatic stage and at the earliest stages of SCA3 neurodegeneration. We therefore assessed plasma NfL and pNfH also in a SCA3 knock-in mouse model (preprint: Haas et al, 2020; Martier et al, 2019) across presymptomatic and symptomatic disease stages, correlating plasma concentrations of both Nfs with the temporal course of phenotypic and neuropathological disease features, including brain ataxin-3 protein levels and aggregation. Results Serum NfL levels are increased at the ataxic stage of SCA3 In cohort #1 (Fig 1A), serum concentrations of NfL were significantly higher in ataxic SCA3 subjects (34.8 pg/ml (28.3–47.0), median and IQR) than in controls (8.6 pg/ml [5.7–11.7]; U = 151, z = 10.1, P < 0.001, r = 0.82). In cohort #2 (Fig 1B), NfL levels were also significantly higher in ataxic SCA3 subjects (85.5 pg/ml [70.2–100.2]) than in controls (19.4 pg/ml [15.1–25.4]; U = 16, z = 6.98, P < 0.001, r = 0.81). This confirmed the NfL increase in a second, independent cohort with an independent immunoassay. NfL levels differentiated between ataxic SCA3 subjects and controls with high accuracy (cohort #1: AUC = 0.97 (0.95–1.00), P < 0.001, optimal cut-off: 20.0 pg/ml, 98.7% sensitivity, 92.2% specificity; cohort #2: AUC = 0.99 (0.97–1.00), P < 0.001, optimal cut-off: 50.9 pg/ml, 92.6% sensitivity, 100% specificity). If corrected for age, the NfL increase in ataxic SCA3 subjects remained highly significant in both cohort #1 (F(1,147) = 406.54, P < 0.001; based on a linear model with the factors group, age and their interaction, R2 = 0.82; Fig 1C) and cohort #2 (F(1,70) = 169.49, P < 0.001, R2 = 0.79; Fig 1D). Figure 1. Serum NfL and pNfH concentrations in the preataxic and ataxic stage of SCA3 A–D. Serum NfL concentrations of preataxic (green) and ataxic (red) SCA3 subjects and controls (blue) were measured in two independent cohorts, each with a different Simoa approach: cohort #1, recruited by the ESMI consortium (A, C), and cohort #2, recruited by the EuroSCA/RiSCA consortium (B, D). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Groups were compared with Mann–Whitney U-tests (***P < 0.001, **P < 0.01, ns P ≥ 0.05, two-tailed, Bonferroni-corrected; see Appendix Table S3 for detailed statistics). In the scatter plots, the individual NfL values were plotted as a function of subjects’ age. The dashed grey lines visualise the optimal cut-offs for differentiating ataxic SCA3 subjects from controls in each cohort (cohort #1: 20.0 pg/ml, 98.7% sensitivity, 92.2% specificity; cohort #2: 50.9 pg/ml, 92.6% sensitivity, 100% specificity; cut-offs were derived by maximising Youden's index irrespective of age). Note the logarithmic scale of the y-axes. E, F. Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts, each with a different Simoa approach (two-tailed Mann–Whitney U-tests, Bonferroni-corrected; see Appendix Table S3 for detailed statistics). Source data are available online for this figure. Source Data for Figure 1 [emmm201911803-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Serum NfL levels are increased at the preataxic stage of SCA3 In cohort #1 (Fig 1A), NfL levels of preataxic SCA3 subjects (29.1 pg/ml [15.9–43.7]) were significantly higher than in controls (U = 72, z = 3.55, P < 0.001, r = 0.39) and did not differ significantly from those of ataxic SCA3 subjects (U = 204, z = 1.48, P = 0.143, r = 0.16, Bonferroni-corrected for multiple comparisons, respectively). In cohort #2 (Fig 1B), NfL levels of preataxic SCA3 subjects (47.3 pg/ml [25.5–78.0]) were also significantly increased (U = 88, z = 4.18, P < 0.001, r = 0.53), yet significantly lower than in ataxic SCA3