Loss of the mitochondrial i ‐ AAA protease YME 1L leads to ocular dysfunction and spinal axonopathy
2018; Springer Nature; Volume: 11; Issue: 1 Linguagem: Inglês
10.15252/emmm.201809288
ISSN1757-4684
AutoresHans‐Georg Sprenger, Gulzar A. Wani, Annika Hesseling, Tim König, Maria Patrón, Thomas MacVicar, Sofia Ahola, Timothy Wai, Esther Barth, Elena I. Rugarli, Matteo Bergami, Thomas Langer,
Tópico(s)Redox biology and oxidative stress
ResumoResearch Article2 November 2018Open Access Source DataTransparent process Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy Hans-Georg Sprenger Hans-Georg Sprenger Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Gulzar Wani Gulzar Wani Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Annika Hesseling Annika Hesseling Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Tim König Tim König Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Maria Patron Maria Patron Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Thomas MacVicar Thomas MacVicar Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Sofia Ahola Sofia Ahola Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Timothy Wai Timothy Wai Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Esther Barth Esther Barth Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Elena I Rugarli Elena I Rugarli Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Matteo Bergami Matteo Bergami Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine, University of Cologne, Cologne, Germany Search for more papers by this author Thomas Langer Corresponding Author Thomas Langer [email protected] orcid.org/0000-0003-1250-1462 Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine, University of Cologne, Cologne, Germany Search for more papers by this author Hans-Georg Sprenger Hans-Georg Sprenger Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Gulzar Wani Gulzar Wani Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Annika Hesseling Annika Hesseling Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Tim König Tim König Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Maria Patron Maria Patron Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Thomas MacVicar Thomas MacVicar Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Sofia Ahola Sofia Ahola Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Timothy Wai Timothy Wai Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Esther Barth Esther Barth Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Elena I Rugarli Elena I Rugarli Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Matteo Bergami Matteo Bergami Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine, University of Cologne, Cologne, Germany Search for more papers by this author Thomas Langer Corresponding Author Thomas Langer [email protected] orcid.org/0000-0003-1250-1462 Max-Planck-Institute for Biology of Ageing, Cologne, Germany Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine, University of Cologne, Cologne, Germany Search for more papers by this author Author Information Hans-Georg Sprenger1,2, Gulzar Wani2, Annika Hesseling2, Tim König2,4, Maria Patron1,2, Thomas MacVicar1,2, Sofia Ahola1,2, Timothy Wai2,5,6, Esther Barth2, Elena I Rugarli2, Matteo Bergami2,3 and Thomas Langer *,1,2,3 1Max-Planck-Institute for Biology of Ageing, Cologne, Germany 2Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 3Center for Molecular Medicine, University of Cologne, Cologne, Germany 4Present address: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada 5Present address: Institut Pasteur, CNRS UMR 3691, Mitochondrial Biology Group, Paris, France 6Present address: Sorbonne Paris Cité, Paris Descartes University, Paris, France *Corresponding author. Tel: +49 221 37 970 500; E-mail: [email protected] EMBO Mol Med (2019)11:e9288https://doi.org/10.15252/emmm.201809288 See also: ZMA Chrzanowska-Lightowlers & RN Lightowlers (January 2019) 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 Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i-AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin-like GTPase OPA1. Mutations in YME1L cause a multi-systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell-type-specific defects in mice lacking YME1L in the nervous system. YME1L-deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects. Synopsis Novel mouse models demonstrate the importance of mitochondrial proteostasis for eye development and axonal maintenance in the spinal cord. Loss of the mitochondrial protease YME1L induces cell-specific neurodegeneration independently of respiratory chain defects and mitochondria fragmentation. Mice lacking YME1L in the nervous system (NYKO mice) show microphthalmia with cataracts and late-onset degeneration of spinal cord axons involved in proprioception. YME1L regulates anterograde axonal transport of mitochondria in cultured neurons. Loss of the mitochondrial protease OMA1 in NYKO mice suppresses aberrant OPA1 processing and mitochondrial fragmentation, but not neurodegeneration in absence of YME1L. OMA1 exerts pro-survival functions and delays the degeneration of YME1L- deficient neurons. Introduction Many prevalent and rare neurodegenerative disorders have been associated with mitochondrial deficiencies, which affect central as well as peripheral parts of the nervous system (Nunnari & Suomalainen, 2012; Kawamata & Manfredi, 2017; Viscomi & Zeviani, 2017; Nissanka & Moraes, 2018). Mitochondria are central metabolic organelles and therefore of pivotal importance for neuronal survival. They provide most neuronal ATP by oxidative phosphorylation (OXPHOS) and thus ensure axonal trafficking of organelles and synaptic transmission. Impaired respiration causes a neuronal energy crisis, inhibits mitochondrial motility, and leads to axonal degeneration in disease, often in a highly neuron-specific manner (Kawamata & Manfredi, 2017; Misgeld & Schwarz, 2017; Viscomi & Zeviani, 2017; Nissanka & Moraes, 2018). Over half of all patients suffering from a mitochondrial disease for instance develop ocular complications, which in some cases are combined with brain atrophy or specific degeneration of motor neurons in the spinal cord (Yu-Wai-Man et al, 2016). The striking cell-type specificity of mitochondrial disorders affecting the nervous system is poorly understood. Similar to OXPHOS defects, deficiencies in other metabolic functions of mitochondria can cause neurodegenerative disorders, as can disturbances in the morphology of mitochondria (Delettre et al, 2000; Chen et al, 2007; Gerber et al, 2017), which is intimately coupled to mitochondrial function and quality control (Youle & van der Bliek, 2012). Dynamin-like GTPases acting on the mitochondrial outer (OM) and inner membrane (IM) ensure the balanced fusion and fission of mitochondria and determine their structure (Friedman & Nunnari, 2014; Mishra & Chan, 2014). Mutations in these GTPases cause complex neurological disorders including dominant optic atrophy (DOA) and Charcot–Marie–Tooth syndrome type 2A (Delettre et al, 2000; Zuchner et al, 2006; Waterham et al, 2007; Gerber et al, 2017). At the level of the IM, the GTPase optic atrophy 1 (OPA1) mediates mitochondrial fusion and regulates cristae morphogenesis (Cogliati et al, 2016). Loss of OPA1 inhibits mitochondrial fusion and results in the fragmentation of the mitochondrial network, which is linked to axonal transport defects and mitophagy (Twig et al, 2008; Gomes et al, 2011). OPA1 undergoes proteolytic processing leading to the balanced accumulation of short (S-) and membrane-anchored long forms (L-) of OPA1 (MacVicar & Langer, 2016). Whereas either L-OPA1 or S-OPA1 is sufficient to preserve cristae structure and respiration (Anand et al, 2014; Del Dotto et al, 2017; Lee et al, 2017), L-OPA1 is required to mediate mitochondrial fusion (Tondera et al, 2009; Ban et al, 2017; Chen & Chan, 2017). Two IM proteases can cleave L-OPA1 at neighboring sites and limit fusion: the stress-activated peptidase OMA1 (Ehses et al, 2009; Head et al, 2009) and the ATP-dependent i-AAA protease YME1L (Griparic et al, 2007; Song et al, 2007). The processing of OPA1 by these two proteases offers additional possibilities of regulation allowing to adapt mitochondrial morphology to distinct physiological cues (Mishra et al, 2014; MacVicar & Langer, 2016). OMA1-deficient mice show impaired OPA1 cleavage and exhibit marked metabolic alterations, highlighting the importance of mitochondrial dynamics for metabolic control (Quiros et al, 2012). The loss of OMA1 impairs the expression of lipid and glucose metabolic enzymes in adipocyte tissues, which results in defective thermoregulation, reduced energy expenditure, and obesity (Quiros et al, 2012). However, the function of OMA1 in the nervous system remains largely elusive. OMA1 activation and mitochondrial fragmentation occurs under various stress conditions, such as mitochondrial depolarization, heat and oxidative stress, and occurs in apoptotic cells (Baker et al, 2014; Jiang et al, 2014; Zhang et al, 2014). Accordingly, OMA1 deficiency reduces the sensitivity to apoptosis (Quiros et al, 2012; Jiang et al, 2014; Korwitz et al, 2016). On the other hand, cells lacking the second OPA1 cleaving peptidase YME1L show an increased vulnerability to apoptosis due to OMA1 activation under these conditions (Stiburek et al, 2012; Anand et al, 2014). YME1L preserves mitochondrial proteostasis and function acting both as a quality control and as a regulatory enzyme in the IM (Quiros et al, 2015; Levytskyy et al, 2017). Besides cleaving OPA1, it degrades damaged or non-assembled IM proteins such as respiratory chain subunits or TIMM17A, a subunit of a protein translocase in the IM (Stiburek et al, 2012; Rainbolt et al, 2013). Moreover, YME1L mediates the turnover of the short-lived lipid transfer proteins PRELID1 and STARD7 in the intermembrane space (IMS) and therefore also regulates mitochondrial phospholipid homeostasis (Potting et al, 2013; Saita et al, 2018). YME1L is essential for embryonic development in mice, whereas YME1L deficiency in adult cardiomyocytes causes dilated cardiomyopathy and heart failure (Wai et al, 2015). The loss of YME1L results in OMA1 activation and mitochondrial fragmentation (Anand et al, 2014; Wai et al, 2015). Cardiac function was maintained upon ablation of Oma1 in these mice, which leads to the accumulation of L-OPA1 and the formation of tubular mitochondria. Thus, imbalanced mitochondrial dynamics is deleterious in the heart and OMA1 promotes cardiomyocyte cell death. Homozygous recessive mutations in human YME1L, which destabilize YME1L and trigger mitochondrial fragmentation, cause a neuromuscular disorder with intellectual disability, motor developmental delay, optic atrophy as well as ataxia and movement deficiencies (Hartmann et al, 2016). However, how YME1L deficiency affects neuronal function remained enigmatic. Here, we show that the loss of YME1L in the nervous system impairs mitochondrial morphology and proteostasis throughout the nervous system but results in striking cell-type-specific neurological defects in mice: While newborn mice show microphthalmia with retinal inflammation and cataracts, spinal cord axons of the dorso-lateral column progressively degenerate with age, impairing coordinated movements. Axonal degeneration correlates with mitochondrial transport defects in YME1L-deficient, cultured neurons. Additional deletion of Oma1 restored mitochondrial morphology in vivo but deteriorated axonal degeneration, suggesting that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes mitochondrial trafficking defects and axonal loss. Results Loss of YME1L in the nervous system causes microphthalmia, cataracts, and retinal inflammation We mated Yme1lfl/fl mice (Wai et al, 2015) with mice expressing the Cre recombinase under the control of the nestin promoter, which is active specifically in neuronal and glial cell precursors (Nestin-Cre) (Tronche et al, 1999). Homozygous offsprings (NYKO mice for Nestin-Yme1l-Knock Out mice) that lack YME1L in the nervous system were born at normal Mendelian ratios. However, newborn NYKO mice showed dramatic microphthalmia and developed cataracts (Fig 1A). The nestin promoter is activated at embryonic day 11 in the nervous system and drives Cre expression in retinal progenitor cells in the optic cup, which differentiate to various retinal cell types starting from embryonic day 12 (MacPherson et al, 2004; Adler & Canto-Soler, 2007). Thus, NYKO mice lack YME1L in all retinal cell types. A histological analysis revealed an overall normal organization of the retina with unaltered thickness of different retinal layers and no dramatic reduction of nuclei in the outer and inner nuclear layer (ONL, INL; Fig 1B, Appendix Fig S1A). The outer plexiform layer (OPL), however, appeared disorganized and contained nuclei that were absent in the OPL of wild-type retinas (Fig 1B and C). Moreover, the accumulation of the glial fibrillary acidic protein (GFAP) indicated neuroinflammation (Fig 1D), which was further substantiated by the increased expression of the proinflammatory cytokines and NF-κB target genes Tnfα, Il-1β, Asc, and Myd88 in retinas of 6-week-old NYKO mice (Fig 1E). These findings are reminiscent of inflammatory responses observed in muscular OPA1 deficiency (Rodriguez-Nuevo et al, 2018). Similar to this model, we observed increased expression of Fgf21 (Fig 1F). However, mtDNA levels remained unaffected in NYKO mice contrasting muscular OPA1 deficiency (Appendix Fig S1B). Figure 1. Loss of YME1L in the nervous system causes microphthalmia, cataracts, and retinal inflammation A. Representative images of eyes and lenses from 6- to 7-week-old wild-type (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars, 5 mm. B. Retinal sagittal cross sections from 6- to 7-week-old mice stained with hematoxylin and eosin. NFL = nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = rods and cones. Scale bars, 30 μm. C. Quantification of nuclei in OPL (area = 1,000 μm2) from retina cross sections of 6- to 7-week-old WT (n = 4) and NYKO (n = 4) mice. D. Immunoblot analysis of retinal lysates from 6- to 7-week-old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control. E. mRNA levels of proinflammatory cytokines and NF-κB target genes from 6- to 7-week-old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. F. mRNA levels of Fgf21 from 6- to 7-week-old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. G. Transmission electron micrographs of optic nerves from 6- to 7-week-old WT and NYKO mice. Scale bars, 2 μm. Data information: Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM. Source data are available online for this figure. Source Data for Figure 1 [emmm201809288-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint We conclude that the loss of YME1L in retinal progenitor cells triggers microphthalmia with cataracts and retinal inflammation. Notably, the analysis of optic nerves in 6-week-old NYKO mice by transmission electron microscopy (TEM) did not reveal axonal degeneration or myelination defects, suggesting that retinal ganglion cells are not broadly affected (Fig 1G and Appendix Fig S1G). Previously identified mitochondrial proteins associated with the development of microphthalmia, namely HCCS and COX7B, were shown to induce a reactive oxygen species (ROS)-dependent induction of caspase 9-mediated cell death (Indrieri et al, 2012, 2013). However, neither the levels of mRNA expression of Hccs or Cox7b, nor the amount of cytochrome c or ROS scavenger enzymes were affected in retinas of NYKO mice (Appendix Fig S1C–E). Moreover, immunoblot analysis of caspase 8 and 9 revealed no activation in retinas deleted for YME1L (Appendix Fig S1F), indicating that the loss of YME1L induces microphthalmia along a different pathway. YME1L ensures locomotor activity and coordinated movements With increasing age, NYKO mice gained less body weight when compared to heterozygous littermates or control mice (Fig 2A). Nuclear magnetic resonance tomography of 31-week-old mice revealed an overall reduced fat mass, with general and subcutaneous white adipose tissue being similarly affected (Figs 2B and EV1A). The whole-body energy expenditure of 26-week-old NYKO mice was significantly increased during the day and night period (Figs 2C and EV1B), although the activity levels of NYKO and control mice were similar (Fig EV1C). The impaired ability of NYKO mice to use the food containers hung at the sensor in the cages prevented a reliable determination of the food intake in these experiments and pointed to locomotor deficiencies of NYKO mice. Figure 2. NYKO mice manifest locomotor impairment of hind limbs A. Mean body weight of male wild-type (WT, n = 15), NYKO (n = 12), and heterozygous NYKO (het NYKO, n = 7) mice from 4 to 32 weeks. B. Body composition analysis by nuclear magnetic resonance (NMR) of 31- to 32-week-old male WT (n = 12) and NYKO (n = 7) mice. C. Whole-body energy expenditure per kg lean mass of 26-week-old male WT (n = 5) and NYKO (n = 5) mice after disease onset. D, E. Walking beam test of 6- to 7-week-old (WT, n = 10; NYKO, n = 10), 17-18-week-old (WT, n = 8; NYKO, n = 9), and 31- to 32-week-old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Hind limb slips per run are shown in (E). F. Representative images of 31- to 32-week-old WT and NYKO mice showing aberrant positioning of hind limbs. Data information: Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Phenotypic characterization of NYKO mice A. Fat pad mass of subcutaneous white adipose tissue (SWAT), gonadal white adipose tissue (GWAT), and brown adipose tissue (BAT) isolated from 31- to 32-week-old WT (n = 12) and NYKO (n = 7) male mice. B. Mean energy expenditure of WT (n = 5) and NYKO (n = 5) male mice after disease onset at 26 weeks of age. C. Day and night activity of 26-week-old, male WT (n = 5) and NYKO (n = 5) mice. D. Wire mesh grip strength test of 6- to 7-week-old WT (n = 10) and NYKO mice (n = 10), 17- to 18-week-old WT (n = 8) and NYKO mice (n = 9), and 31- to 32-week-old mice WT (n = 12), NYKO (n = 19), and heterozygous NYKO mice (het NYKO, n = 9). Data information: Unpaired t-test was used for comparison of two groups, ordinary one-way ANOVA for comparison of three groups. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM. Download figure Download PowerPoint We therefore performed walking beam tests and monitored the time needed for the mice to traverse the beam (Fig 2D). NYKO mice were phenotypically normal at 6 weeks but showed a progressive locomotor impairment starting at 17 weeks of age (Fig 2D). Similarly, we observed an increasing number of slips per run, which was pronounced in 31-week-old NYKO mice shortly before we had to sacrifice the animals for ethical reasons. These locomotor deficiencies were absent in heterozygous animals excluding deleterious effects of the presence of the Cre recombinase (Fig 2D and E). Remarkably, this progressive degenerative phenotype was restricted to the hind limbs, which were positioned abnormally (Fig 2F, Movies EV1 and EV2). In agreement with these observations, grip strength tests revealed that NYKO mice but not heterozygous littermates developed a dramatic impairment in their grip strength at 31 weeks, with their hind limbs being primarily affected (Fig EV1D). We therefore conclude that the absence of YME1L in the mouse nervous system causes a late-onset progressive locomotor impairment of the hind limbs. Neuroinflammation and axonal degeneration in spinal cord of NYKO mice Locomotor control depends on the integration of neurons in the brain. An impaired locomotor activity correlates with brain atrophy and inflammation in many mitochondrial diseases (Maltecca et al, 2009; Almajan et al, 2012; Johnson et al, 2013; Ignatenko et al, 2018). We therefore analyzed brain morphology and brain weight in NYKO mice. Unexpectedly, brains of NYKO mice were morphologically normal and showed no signs of atrophy up to 31 weeks of age (Fig 3A). Nissl and Calbindin stainings of different brain areas of NYKO mice were indistinguishable from those of control mice (Fig 3B and C and Appendix Fig S2A). Moreover, loss of YME1L in the brain did not lead to neuroinflammation. We did not observe the accumulation of neuroinflammatory markers, such as GFAP and ionized calcium binding adaptor molecule 1 (IBA1), upon immunohistochemical and immunoblot analysis of brain tissue from 31-week-old NYKO mice (Fig 3D and Appendix Fig S2A). The pro-inflammatory cytokines Tnfα, Il-6, and Il-1β were expressed at similar levels in NYKO and control mice (Appendix Fig S2B). These results exclude general brain atrophy as the cause for the impaired locomotor activity of aged NYKO mice. Figure 3. The loss of YME1L in the nervous system does not trigger brain atrophy A. Brain weights were monitored at 31–32 weeks of age (WT, n = 15; NYKO, n = 14). B. Nissl stainings of sagittal sections across brain regions from 31- to 32-week-old WT and NYKO mice. 4× and 40× enlargements are shown in the left and right panels, respectively. Scale bars, 4× = 500 μm, 40× = 50 μm. C. Morphometric analysis of different brain regions from 31- to 32-week-old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = molecular layer. D. Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31- to 32-week-old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Data information: Unpaired t-test, ns = not significant. Data are means ± SEM. Source data are available online for this figure. Source Data for Figure 3 [emmm201809288-sup-0008-SDataFig3.pdf] Download figure Download PowerPoint Coordinated movement also relies on the integrity of neurons in the spinal cord (Fig 4A). We therefore examined spinal cords of NYKO mice in further experiments. Transverse spinal cord sections were stained with toluidine blue, which allows detection of myelin dense bodies (MDBs). MDBs are a hallmark of axonal degeneration, as they accumulate when the myelin sheath collapses into the area formerly occupied by the axon (Jeong et al, 2011). Non-symptomatic 6-week-old NYKO mice did not show any morphological abnormalities in the white matter of the spinal cord (Fig 4B and C). MDBs were not detected in the different tracts of the spinal cord. Moreover, we did not observe changes in myelination by TEM (Fig EV2A and B). However, MDBs accumulated in the dorso-lateral tracts of the spinal cord of 17-week-old NYKO mice (Fig 4B and C). They formed more prominently in the cervical part of the spinal cord than in the lumbar part, whereas in aged animals, they were present to the same extent in both parts (Fig 4B and C). Strikingly, axons of the dorso-lateral tract were predominantly affected by the loss of YME1L. We observed only small numbers of MDBs in other ventral and ventro-lateral tracts of aged NYKO mice (Fig EV2C and D). In agreement with the degeneration of axons in the spinal cord, we observed upregulation of GFAP and increased expression of proinflammatory cytokines in the spinal cord of 31-week-old NYKO mice, indicating neuroinflammation (Fig 4D and E). Figure 4. Progressive axonal degeneration of dorso-lateral tracts and neuroinflammation in NYKO spinal cords A. Spinal cord tracts anatomy, transverse section stained with toluidine blue. 1 = dorso-lateral tracts (ascending pathways), 2 = ventro-lateral tracts (ascending pathways), 3 = ventral tracts (descending pathways). B. Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age. Sections were stained with toluidine blue. Orange arrows indicate degenerating neurons (myelin dense bodies, MDBs). Scale bars, 25 μm. C. Myelin dense bodies (MDBs) in dorso-lateral tracts of 6- to 7-week-old WT (n = 4) and NYKO (n = 4), 17- to 18-week-old WT (n = 4) and NYKO (n = 4), and 31- to 32-week-old WT (n = 6–7) and NYKO (n = 5–6) mice spinal cords. D. Immunoblots of 31- to 32-week-old spinal cord lysates. GAPDH was used to control for gel loading. E. mRNA levels of p
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