Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1‐dependent TFEB/TFE3 regulation
2021; Springer Nature; Volume: 13; Issue: 5 Linguagem: Inglês
10.15252/emmm.202013258
ISSN1757-4684
AutoresReini E.N. van der Welle, Rebekah Jobling, Christian Burns, Paolo Sanzà, Jan van der Beek, Alfonso Fasano, Lan Chen, Fried Zwartkruis, Susan Zwakenberg, Edward F. Griffin, Corlinda ten Brink, Tineke Veenendaal, Nalan Liv, Conny M.A. van Ravenswaaij‐Arts, Henny H. Lemmink, Rolph Pfundt, Susan Blasér, Carolina Sepulveda, Andrés M. Lozano, Grace Yoon, Teresa Santiago‐Sim, Cédric S. Asensio, Guy A. Caldwell, Kim A. Caldwell, David Chitayat, Judith Klumperman,
Tópico(s)Cellular transport and secretion
ResumoArticle14 April 2021Open Access Source DataTransparent process Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation Reini E N van der Welle orcid.org/0000-0002-5591-2846 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Rebekah Jobling Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Search for more papers by this author Christian Burns Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Paolo Sanza Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jan A van der Beek orcid.org/0000-0003-4957-9175 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Alfonso Fasano Edmond J. Safra Program in Parkinson's Disease, Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, UHN, Toronto, ON, Canada Division of Neurology, University of Toronto, Toronto, ON, Canada Krembil Brain Institute, Toronto, ON, Canada Center for Advancing Neurotechnological Innovation to Application (CRANIA), Toronto, ON, Canada Search for more papers by this author Lan Chen Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Fried J Zwartkruis orcid.org/0000-0001-5775-1313 Section Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Susan Zwakenberg Section Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Edward F Griffin Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author Corlinda ten Brink Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Tineke Veenendaal Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Nalan Liv Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Conny M A van Ravenswaaij-Arts Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Henny H Lemmink orcid.org/0000-0002-2748-803X Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Rolph Pfundt Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Susan Blaser Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Carolina Sepulveda Edmond J. Safra Program in Parkinson's Disease, Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, UHN, Toronto, ON, Canada Division of Neurology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andres M Lozano Krembil Brain Institute, Toronto, ON, Canada Center for Advancing Neurotechnological Innovation to Application (CRANIA), Toronto, ON, Canada Department of Neurosurgery, Toronto Western Hospital, UHN, Toronto, ON, Canada University of Toronto, Toronto, ON, Canada Search for more papers by this author Grace Yoon Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Search for more papers by this author Teresa Santiago-Sim GeneDx, Inc, Gaithersburg, MD, USA Search for more papers by this author Cedric S Asensio orcid.org/0000-0002-2284-0217 Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Guy A Caldwell orcid.org/0000-0002-8283-9090 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author Kim A Caldwell orcid.org/0000-0003-1580-6122 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author David Chitayat Corresponding Author [email protected] orcid.org/0000-0003-1944-7294 Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Judith Klumperman Corresponding Author [email protected] orcid.org/0000-0003-4835-6228 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Reini E N van der Welle orcid.org/0000-0002-5591-2846 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Rebekah Jobling Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Search for more papers by this author Christian Burns Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Paolo Sanza Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jan A van der Beek orcid.org/0000-0003-4957-9175 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Alfonso Fasano Edmond J. Safra Program in Parkinson's Disease, Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, UHN, Toronto, ON, Canada Division of Neurology, University of Toronto, Toronto, ON, Canada Krembil Brain Institute, Toronto, ON, Canada Center for Advancing Neurotechnological Innovation to Application (CRANIA), Toronto, ON, Canada Search for more papers by this author Lan Chen Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Fried J Zwartkruis orcid.org/0000-0001-5775-1313 Section Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Susan Zwakenberg Section Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Edward F Griffin Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author Corlinda ten Brink Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Tineke Veenendaal Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Nalan Liv Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Conny M A van Ravenswaaij-Arts Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Henny H Lemmink orcid.org/0000-0002-2748-803X Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Rolph Pfundt Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Susan Blaser Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Carolina Sepulveda Edmond J. Safra Program in Parkinson's Disease, Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, UHN, Toronto, ON, Canada Division of Neurology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andres M Lozano Krembil Brain Institute, Toronto, ON, Canada Center for Advancing Neurotechnological Innovation to Application (CRANIA), Toronto, ON, Canada Department of Neurosurgery, Toronto Western Hospital, UHN, Toronto, ON, Canada University of Toronto, Toronto, ON, Canada Search for more papers by this author Grace Yoon Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Search for more papers by this author Teresa Santiago-Sim GeneDx, Inc, Gaithersburg, MD, USA Search for more papers by this author Cedric S Asensio orcid.org/0000-0002-2284-0217 Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA Search for more papers by this author Guy A Caldwell orcid.org/0000-0002-8283-9090 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author Kim A Caldwell orcid.org/0000-0003-1580-6122 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Search for more papers by this author David Chitayat Corresponding Author [email protected] orcid.org/0000-0003-1944-7294 Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Judith Klumperman Corresponding Author [email protected] orcid.org/0000-0003-4835-6228 Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Author Information Reini E N van der Welle1, Rebekah Jobling2, Christian Burns3, Paolo Sanza1, Jan A van der Beek1, Alfonso Fasano4,5,6,7, Lan Chen3, Fried J Zwartkruis8, Susan Zwakenberg8, Edward F Griffin9,10, Corlinda ten Brink1, Tineke Veenendaal1, Nalan Liv1, Conny M A van Ravenswaaij-Arts11, Henny H Lemmink11, Rolph Pfundt12, Susan Blaser13, Carolina Sepulveda4,5, Andres M Lozano6,7,14,15, Grace Yoon2, Teresa Santiago-Sim16, Cedric S Asensio3, Guy A Caldwell9,10, Kim A Caldwell9,10, David Chitayat *,2,17 and Judith Klumperman *,1 1Section Cell Biology, Center for Molecular Medicine, Institute of Biomembranes, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands 2Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada 3Department of Biological Sciences, Division of Natural Sciences and Mathematics, University of Denver, Denver, CO, USA 4Edmond J. Safra Program in Parkinson's Disease, Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, UHN, Toronto, ON, Canada 5Division of Neurology, University of Toronto, Toronto, ON, Canada 6Krembil Brain Institute, Toronto, ON, Canada 7Center for Advancing Neurotechnological Innovation to Application (CRANIA), Toronto, ON, Canada 8Section Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands 9Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA 10Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Nathan Shock Center for Basic Research in the Biology of Aging, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA 11Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 12Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 13Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, ON, Canada 14Department of Neurosurgery, Toronto Western Hospital, UHN, Toronto, ON, Canada 15University of Toronto, Toronto, ON, Canada 16GeneDx, Inc, Gaithersburg, MD, USA 17The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada *Corresponding author. Tel: +1 416 586 4523; E-mail: [email protected] *Corresponding author. Tel: +31 88 7556550; E-mail: [email protected] EMBO Mol Med (2021)13:e13258https://doi.org/10.15252/emmm.202013258 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 Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662*; VPS41c.1423-2A>G and VPS41R662*) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P/VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease. Synopsis Compound heterozygous mutations in VPS41 were identified in patients with a neurodegenerative phenotype with dystonia and cerebellar atrophy. VPS41 variants obstruct the HOPS complex, leading to decreased endocytic and autophagy cargo transfer to lysosomes and inhibition of mTORC1 signaling. VPS41 variants lead to an inhibition of mTORC1-mediated TFE3/TFEB regulation, whereas S6K1/4EBP1/ULK1 remain unaffected. Depletion of HOPS complex subunits prevents TFE3/TFEB phosphorylation, indicating a regulatory role of the HOPS complex in the TFE3/TFEB axis of mTORC1 signaling. Depletion of VPS41 or other HOPS components results in a constitutive upregulation of autophagy and a failure to respond to nutrient starvation. VPS41 variant S285P blocks HOPS complex formation but allows regulated secretion. VPS41 variants decrease the neuroprotective effect of VPS41 against α-synuclein overexpression in dopaminergic neurons. The paper explained Problem VPS41 is, as part of the HOPS complex, required for lysosomal fusion events. Independently of HOPS, VPS41 is required for formation of secretory granules. VPS41 prevents degeneration of dopaminergic neurons overexpressing the Parkinson's disease-related protein α-synuclein. Here, we present three patients bearing compound heterozygote mutations in VPS41 displaying a severe neurological disorder. We address the question how these mutations affect endocytosis, autophagy, secretory pathways, and neuroprotection. Results We show that mutations in or depletion of VPS41 causes a delay in HOPS-dependent endocytic and autophagic cargo delivery to enzymatically active lysosomes and that neuroprotection against α-synuclein is reduced. Moreover, the disease-causing mutations specifically impair mTORC1 activity toward TFE3 and TFEB, causing constitutive activation of these transcription factors. As a result, lysosomal biogenesis and autophagosome formation is continuously upregulated independent of nutrient conditions. By contrast, HOPS-independent function of VPS41 in secretory transport is preserved. Impact Our study shows that VPS41 patients represent a new class of lysosomal disorders in which lysosomes are functional, but insufficiently reached by cargo due to a trafficking defect. Besides this, we show that VPS41, as part of the HOPS complex, is involved in the differential regulation of mTORC1 toward TFE3/TFEB, which is of potential importance for treatment of HOPS-related disorders. Introduction While lysosomes are responsible for the degradation and recycling of intra- and extracellular substrates (Saftig & Klumperman, 2009), they are increasingly recognized as regulators of cellular homeostasis and key players in nutrient sensing and transcriptional regulation (Luzio et al, 2007; Richardson et al, 2010; Settembre et al, 2015; Mony et al, 2016). Hence, the digestive properties of lysosomes provide them with a major role in the control of cellular metabolism and nutrient homeostasis. To accomplish this, HOPS (Homotypic Fusion and Protein Sorting), a multisubunit tethering complex, regulates fusion of lysosomes with endosomes and autophagosomes (Seals et al, 2000; Wurmser et al, 2000; Caplan et al, 2001; Pols et al, 2013a; Kant et al, 2015; Beek et al, 2019). Vacuolar Protein Sorting 41 (VPS41) (VPS41, found on Chromosomal location; Chr7p14.1) is a defining component of HOPS, and a 100 kD protein that contains a WD40, TRP-like (tetratricopeptide repeat), CHCR (Clathrin Heavy Chain Repeat), and RING (Really Interesting New Gene)-H2 Zinc Finger domain (Radisky et al, 1997; Nickerson et al, 2009). VPS41 knockout mice die early in utero, signifying VPS41 as an essential protein for embryonic development (Aoyama et al, 2012). HOPS-associated VPS41 is recruited to endosomes by binding to Rab7 and its interactor Rab interacting lysosomal protein, and to lysosomes by interacting with Arf-like protein 8b (Arl8b; Lin et al, 2014; Khatter et al, 2015). Depletion of VPS41 impairs HOPS-dependent delivery of endocytic cargo to lysosomes and causes a defect in autophagic flux (Takáts et al, 2009; Pols et al, 2013a). Independent of HOPS, VPS41 is required for transport of lysosomal membrane proteins from the trans-Golgi-Network (TGN) to lysosomes (Swetha et al, 2011; Pols et al, 2013b), akin to the Alkaline Phosphatase (ALP) pathway in yeast (Cowles et al, 1997a; Rehling et al, 1999; Darsow et al, 2001; Angers & Merz, 2009; Cabrera et al, 2010). Furthermore, in secretory cells and neurons, VPS41 is required for regulated secretion of neuropeptides (Burgess & Kelly, 1987; Orci et al, 1987; Tooze & Huttner, 1990; Eaton et al, 2000; Asensio et al, 2010; Asensio et al, 2013; Hummer et al, 2017). Together, these data show that VPS41, as part of HOPS, is required for lysosomal fusion events and, independent of HOPS, for transport of lysosomal membrane proteins and regulated secretion. Interestingly, VPS41 overexpression protects dopaminergic neurons against neurodegeneration. This was shown in a transgenic C. elegans model of Parkinson's disease, in which α-synuclein is locally overexpressed (Hamamichi et al, 2008; Ruan et al, 2010; Harrington et al, 2012), and in human neuroglioma cells overexpressing α-synuclein (Harrington et al, 2012). Neuroprotection against α-synuclein requires interaction of VPS41 with Rab7 and adaptor protein-3 (AP-3) (Griffin et al, 2018). Recent studies in C. elegans showed that overexpression of human VPS41 also mitigates Aβ-induced neurodegeneration of glutamatergic neurons. This requires the small GTPase Arl8b rather than Rab7 or AP-3, indicating that VPS41, through different interaction partners, can trigger divergent neuroprotective mechanisms against Parkinson's disease and Alzheimer's disease (Griffin et al, 2018). However, how VPS41's function leads to neuroprotection remains to be elucidated. Naturally occurring SNPs in VPS41 have been described (T52R, T146P and A187T; Harrington et al, 2012; Ibarrola-Villava et al, 2015), and very recently a single patient with early onset dystonia and a homozygous canonical splice site variant in VPS41 was identified (Steel et al, 2020). In this patient, cDNA studies demonstrated that the variant leads to in-frame skipping of exon 7. In our current study, we present three patients with severe neurological features (e.g., ataxia and dystonia accompanied by retinal dystrophy and mental retardation with brain MRI findings of cerebellar atrophy and thin corpus callosum) of unknown etiology. Exome sequencing showed that the patients were compound heterozygous for variants in VPS41 [NM_014396.19: c.853T > C, NP_055211.2: p. Ser285Pro (S285P); NM_014396.6: c.1984C > T, NP_055211.2: p. Arg662Stop (R662*); NM_014396.3: c.1423-2A > G, r.(spl?)]. At the cellular level, we show that expression of these VPS41 variants prevents the formation of a functional HOPS complex, leading to a kinetic defect in the delivery of endocytosed and autophagic cargo to lysosomes. In addition, we find an inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) toward TFEB/TFE3 and concomitantly a constitutive high level of autophagy with a failure to respond to changing nutrient conditions. Finally, we show that compound expression of VPS41S285P and VPS41R662* abolishes the neuroprotective effect of VPS41 in the C. elegans model of Parkinson's disease. To the best of our knowledge, this is the first study on the cellular consequences of biallelic VPS41 variants in patients displaying neurological manifestations. Our molecular analysis shows that these mutations result in an unexpected defect in mTORC1 signaling, specifically in the TFEB/TFE3 axis regulating autophagy. Patients Clinical presentation of three patients with biallelic variants in VPS41 Family 1 (Fig EV1A)—Patients 1 and 2, two brothers, born via spontaneous and vaginal delivery, at term, to healthy and non-consanguineous parents. Their birth weights were 3.03 kg (10th–50th centile) and 3.34 kg (10th–50th centile), respectively. The birth length and head circumferences were not recorded. No abnormalities were noted after birth, and the babies were discharged on time. Both presented with neonatal hypotonia and poor eye contact and fixation at 2 months of age and both had ophthalmological examination showing hypopigmented and underdeveloped retina which disappeared at 1 year of age. Physical examination at this stage showed a high forehead with frontal bossing, retrognathia, deep set eyes, short and pointed nose, and prominent ears. There was no organomegaly. In infancy, they were noted to have global developmental delay, poor muscle tone, and marked intentional tremor which further impaired their fine and gross motor skills. Both brothers developed progressive spasticity of the lower limbs with some coarsening of the facial features more so in patient 2 with puffy eyelids, heavy eyebrows, and thick lips. Both brothers showed absent deep tendon reflexes (DTRs), and their plantars were extensor. Both developed upper extremity tremor and significant lower limbs' spasticity and ataxia (Movies EV1–EV3). Extensive investigation for metabolic diseases, including lysosomal and mitochondrial disorders, showed no detectable abnormalities. No urinary mucopolysaccharides and oligosaccharides were detected. Cerebrospinal fluid analysis for neurotransmitter levels showed no abnormalities. Brain MRI done in infancy on both brothers showed mild hypomyelination (Fig 1A and B). However, a repeat brain MRI study on the older brother (patient 1) at 4 years 7 months showed thin corpus callosum with a saber-shape configuration (Fig 1A) and the vermis, although normal in configuration and size initially, demonstrated volume loss on follow-up examinations. Furthermore, brain MRI done on the younger brother (patient 2) at 10 years of age confirmed the thin corpus callosum, mild progression of the cerebellar atrophy, bilateral hypointensity in the globus pallidus (GP) compatible with early iron deposition, and abnormal hyperintensity in the dentate nucleus (Fig 1B). The overall findings were consistent with neurodegenerative rather than solely malformative brain disease. The growth charts for head circumference were within normal range (Fig EV1B). Click here to expand this figure. Figure EV1. Mutations in VPS41 cause a neurodegenerative disease Pedigrees of family 1 affected by recessive mutations in VPS41. Circle = female, square = male, black-filled shape = individual phenotypically affected. Head circumference of patients 1 (green), 2 (yellow), and 3 (red) in cm. Outline of the VPS41 protein depicting distinct domains. The WD40 domain facilitates protein–protein interactions and the CHCR and RING domains enable homo-oligomerization and are required for HOPS complex formation and regulated secretion. Mutations in VPS41 were identified using whole exome sequencing. The two siblings (patients 1 and 2) bear compound heterozygous mutations; a missense mutation VPS41S285P in the WD40 domain and a nonsense mutation VPS41R662* at the C-terminus resulting in a premature stop codon. Patient 3 bears a canonical splicing variant expected to destroy an acceptor site (VPS41c.1423-2A>G) in the TPR-like domain and shares the nonsense mutation (VPS41R662*) at the C terminus with the other patients. Pedigree of family 2 affected by recessive mutations in VPS41. Circle = female, square = male, black-filled shape = individual phenotypically affected, double line = consanguinity. Patient 3 developed coarse facial features with heavy eyebrows, gingival hypertrophy, protruding tongue, thick lips, and thick ear lobes (I; age 1 year, 7 months, II and III; age 4 years, 10 months). A spinal X-ray showed a kyphosis at C3 and hypoplastic distal phalanges on digits 2, 3, and 5 and a short metacarpal 1, bilaterally (IV; age 5 years, V; age 11 years). Download figure Download PowerPoint Figure 1. Mutations in VPS41 cause a neurodegenerative disease Patient 1 (older sibling) underwent 3 MRI studies, first and last are shown. Top row: 21 months; Bottom row: 4 years 7 months. The corpus callosum is thin on T1-weighted sagittal images (I, V, VI) with a saber shape (long arrows). The shape remains consistent over time. The vermis is normal in configuration and size initially (I), but demonstrates volume loss on follow-up (V, VI) examination (short arrows). FLAIR axial image (II) is age appropriate, while (VII) demonstrates periatrial increased signal (arrow). There is delay in myelin maturation present on T1 (III, VIII)- and T2 (IV, IX)-weighted axial images (long arrows) on initial examination, with further slow myelin development over time. There is deficiency of periatrial white matter volume on both T1 (III, VIII)- and T2 (IV, IX)-weighted axial images (short arrows). C
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