Distinct roles of α‐ and β‐tubulin polyglutamylation in controlling axonal transport and in neurodegeneration
2021; Springer Nature; Volume: 40; Issue: 17 Linguagem: Inglês
10.15252/embj.2021108498
ISSN1460-2075
AutoresSatish Bodakuntla, Xidi Yuan, Mariya Genova, Sudarshan Gadadhar, Sophie Leboucher, Marie‐Christine Birling, Dennis Klein, Rudolf Martini, Carsten Janke, Maria M. Magiera,
Tópico(s)Histone Deacetylase Inhibitors Research
ResumoArticle26 July 2021free access Source DataTransparent process Distinct roles of α- and β-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration Satish Bodakuntla Satish Bodakuntla orcid.org/0000-0002-0448-7683 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Xidi Yuan Xidi Yuan Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Mariya Genova Mariya Genova Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Sudarshan Gadadhar Sudarshan Gadadhar orcid.org/0000-0003-4791-2034 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Sophie Leboucher Sophie Leboucher Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Marie-Christine Birling Marie-Christine Birling orcid.org/0000-0002-3372-8108 CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, Illkirch, France Search for more papers by this author Dennis Klein Dennis Klein Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Rudolf Martini Rudolf Martini orcid.org/0000-0002-5463-5592 Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Carsten Janke Corresponding Author Carsten Janke [email protected] orcid.org/0000-0001-7053-2000 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Maria M Magiera Corresponding Author Maria M Magiera [email protected] orcid.org/0000-0003-4847-3053 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Satish Bodakuntla Satish Bodakuntla orcid.org/0000-0002-0448-7683 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Xidi Yuan Xidi Yuan Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Mariya Genova Mariya Genova Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Sudarshan Gadadhar Sudarshan Gadadhar orcid.org/0000-0003-4791-2034 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Sophie Leboucher Sophie Leboucher Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Marie-Christine Birling Marie-Christine Birling orcid.org/0000-0002-3372-8108 CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, Illkirch, France Search for more papers by this author Dennis Klein Dennis Klein Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Rudolf Martini Rudolf Martini orcid.org/0000-0002-5463-5592 Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Carsten Janke Corresponding Author Carsten Janke [email protected] orcid.org/0000-0001-7053-2000 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Maria M Magiera Corresponding Author Maria M Magiera [email protected] orcid.org/0000-0003-4847-3053 Institut Curie, Université PSL, CNRS UMR3348, Orsay, France Université Paris-Saclay, CNRS UMR3348, Orsay, France Search for more papers by this author Author Information Satish Bodakuntla1,2,5, Xidi Yuan3, Mariya Genova1,2, Sudarshan Gadadhar1,2, Sophie Leboucher1,2, Marie-Christine Birling4, Dennis Klein3, Rudolf Martini3, Carsten Janke *,1,2 and Maria M Magiera *,1,2 1Institut Curie, Université PSL, CNRS UMR3348, Orsay, France 2Université Paris-Saclay, CNRS UMR3348, Orsay, France 3Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Würzburg, Germany 4CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, Illkirch, France 5Present address: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA *Corresponding author. Tel. +33 1 69863127; E-mail: [email protected] *Corresponding author. Tel. +33 1 69863088; E-mail: [email protected] The EMBO Journal (2021)40:e108498https://doi.org/10.15252/embj.2021108498 Correction(s) for this article Distinct roles of α- and β-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration01 June 2022 Correction added on 1 June 2022, after first online publication: Panel E has been corrected. See the associated Corrigendum at https://doi.org/10.15252/embj.2022111373 Correction added on 1 June 2022, after first online publication: Figure 5 has been corrected. See the associated Corrigendum at https://doi.org/10.15252/embj.2022111373 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 Tubulin polyglutamylation is a post-translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The "tubulin code" hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation-specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α-tubulin, while TTLL7 modifies β-tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis. SYNOPSIS Polyglutamylation is a posttranslational modification of tubulin that is highly enriched in neurons. Here we demonstrate that two neuronal polyglutamylases, TTLL1 and TTLL7, have distinct enzymatic activities, which generate unique patterns of polyglutamylation in vivo. We find that TTLL1, but not TTLL7 affects mitochondria transport and neuronal survival, in both central and peripheral nervous system. TTLL1 polyglutamylates α-tubulin, while TTLL7 modifies β-tubulin in vivo. In the absence of the deglutamylase CCP1, excessive polyglutamylation leading to neurodegeneration is generated by TTLL1, but not by TTLL7. Degeneration of neurons in both, central and peripheral nervous system, can be avoided by inactivating TTLL1, but not TTLL7. Polyglutamylation generated by TTLL1, but not by TTLL7 affects mitochondria mobility in axons of hippocampal neurons. Introduction Given the multitude of essential functions of the microtubule cytoskeleton in neurons, it is no surprise that a large number of neurodegenerative disorders involve dysfunctions of microtubules and their associated proteins (reviewed in Matamoros & Baas, 2016). Surprisingly though, little is known on the cytoskeletal functions that might be perturbed in these disorders. The search for pathological mechanisms is difficult as microtubules in neurons have a variety of essential roles, such as determination of neuron shape (Baas et al, 2016) and polarity (van Beuningen & Hoogenraad, 2016), control of intra-neuronal transport (Guedes-Dias & Holzbaur, 2019; Sleigh et al, 2019) and synaptic activity (Guedes-Dias & Holzbaur, 2019; Dent, 2020), and they are also involved in neuronal regeneration (Blanquie & Bradke, 2018). How these distinct functions can be coordinated within one and the same microtubule cytoskeleton mostly remained a conundrum. Understanding how distinct microtubule functions can be selectively controlled in neurons might provide a key to understand cytoskeletal alterations in neurodegenerative diseases. The concept of the tubulin code (Verhey & Gaertig, 2007) proposes that microtubule properties and functions can be determined by tubulin isotypes incorporated into the microtubules and/or by modifying microtubules with a set of characteristic post-translational modifications (PTMs) (Janke & Magiera, 2020). Tubulin PTMs can rapidly and dynamically change the identity of microtubules in cells and might thereby "encode" them for specific functions. Indeed, microtubules in neurons carry many of the well-characterised tubulin PTMs, indicating that they play key roles in coordinating neuronal microtubule functions (Janke & Magiera, 2020). One PTM that is strongly enriched in neurons is polyglutamylation, a polymodification that adds secondary peptide chains of variable numbers of glutamates onto glutamic acid residues of the primary peptide chains of tubulins. All currently known sites of polyglutamylation are located within the carboxy-terminal (C-terminal) tails of α- and β-tubulin (Eddé et al, 1990; Alexander et al, 1991; Rüdiger et al, 1992). Polyglutamylation is catalysed by a variety of enzymes from the tubulin tyrosine ligase-like (TTLL) family (Janke et al, 2005; van Dijk et al, 2007) and is reversed by deglutamylases from the cytosolic carboxypeptidase (CCP) family (Rogowski et al, 2010; Tort et al, 2014). Each glutamylase has a characteristic way to modify tubulin: enzymes have preferences for either α- or β-tubulin, as well as for the initiation or elongation of the glutamate chains (van Dijk et al, 2007; Mahalingan et al, 2020). Henceforth, it is expected that each of these enzymes generates a unique polyglutamylation pattern on microtubules, which in turn "encodes" specific microtubule functions. However so far, this hypothesis has not been tested under physiological conditions. In neurons, several glutamylases and deglutamylases are expressed. Strikingly, and in stark contrast to tubulin-modifying enzymes involved in acetylation and detyrosination, two other tubulin PTMs that are also prominent in neurons, enzymes catalysing polyglutamylation are predominantly expressed in the adult brain (Bodakuntla et al, 2020b), suggesting that polyglutamylation might play a central role in neuronal homeostasis throughout life. This notion has been confirmed by our recent findings that the lack of deglutamylating enzymes, which causes an abnormal increase of polyglutamylation, leads to neurodegeneration in mice and humans (Rogowski et al, 2010; Magiera et al, 2018; Shashi et al, 2018). By contrast, mouse models in which other tubulin PTMs were altered present either early neurodevelopmental abnormalities as seen in a mouse with strongly increased tubulin detyrosination (Erck et al, 2005; Aillaud et al, 2017) or no striking neuronal phenotypes, such as a mouse lacking tubulin acetylation (Kalebic et al, 2013). The causative role of abnormally accumulated polyglutamylation in neurodegeneration has initially been demonstrated in the so-called Purkinje-cell-degeneration (pcd) mice (Mullen et al, 1976) that carry a mutation in the AGTPBP1 or NNA1 gene (Fernandez-Gonzalez et al, 2002) encoding the deglutamylase CCP1. Loss of CCP1 in these mice leads to hyperglutamylation in brain regions where CCP1 is the main deglutamylase (Rogowski et al, 2010) and which had been shown to degenerate in pcd mice (Mullen et al, 1976; Greer & Shepherd, 1982). The degeneration of the Purkinje cells in the cerebellum of CCP1-deficient mice could be entirely prevented by the concomitant depletion of the polyglutamylase TTLL1 (Berezniuk et al, 2012; Magiera et al, 2018), which suggested that re-equilibration of polyglutamylation saved these neurons from degeneration. While this demonstrated that accumulation of polyglutamylation is causative for the observed degeneration, the underlying mechanisms of these defects have not been fully understood. Increased polyglutamylation led to a reduction of axonal transport of a variety of neuronal cargoes in hippocampal pyramidal neurons, with a particularly strong impact on mitochondria motility (Magiera et al, 2018; Bodakuntla et al, 2020b), which was also found in cerebellar granule neurons (Gilmore-Hall et al, 2019). How tightly this transport defect is linked to different polyglutamylation levels on neuronal microtubules and whether specific polyglutamylases play distinct roles in controlling neuronal transport and causing neurodegeneration have remained open questions. Here, we generated a series of a novel mouse models lacking combinations of the deglutamylase CCP1 and glutamylases TTLL1 (Janke et al, 2005) and TTLL7 (Ikegami et al, 2006) to demonstrate that different patterns of polyglutamylation generated by specific enzymes have distinct biological functions in controlling axonal transport and the overall survival of different neurons in vivo. Results TTLL1 and TTLL7 are the two main glutamylases in adult mouse brain Our recent expression analysis of tubulin-modifying enzymes in brain tissue had shown that several TTLL family members are expressed in adult brain. Using quantitative RT–PCR, we found substantial expression levels for the glutamylases TTLL1, TTLL5, TTLL7 and TTLL11 (Bodakuntla et al, 2020b). Previous biochemical work has shown that TTLL1 is a major polyglutamylase in brain tissue, as depletion of this enzyme led to a massive loss of the overall glutamylation activity in brain extracts (Janke et al, 2005). A second enzyme with an important role in the nervous system is TTLL7, as depletion of this enzyme in cultured neuronal cells caused a drop in β-tubulin glutamylation (Ikegami et al, 2006). These early experiments suggested that TTLL1 and TTLL7 might be key enzymes involved in the control of tubulin polyglutamylation in adult brain; however, functional evidence for this hypothesis was so far lacking. To determine the contribution of TTLL1 and TTLL7 to microtubule polyglutamylation in the nervous system, we first characterised mouse strains lacking either of these two enzymes. While the Ttll1−/− mouse was already available (Magiera et al, 2018), we generated a new Ttll7−/− mouse model for this study (Appendix Fig S1). We first characterised polyglutamylation status in brain tissue of these two mouse models using a panel of (poly)glutamylation-specific antibodies. As the established antibodies GT335, recognising the branch point of glutamate chains (Wolff et al, 1992), and polyE, which specifically detects long glutamate chains, and thus polyglutamylation (Shang et al, 2002; Rogowski et al, 2010), barely detected β-tubulin glutamylation, we developed a novel antibody exclusively recognising β-tubulin glutamylation. Early mass-spectrometry analyses had pinpointed that the major β-tubulin isotype in the brain, β2-tubulin, is mostly glutamylated on the glutamate residue (E) 435 (Rüdiger et al, 1992). This glutamylation site is preceded by a glycine (G) and followed by a phenylalanine (F) residue (Fig 1A) and can therefore not be detected by the broadly used antibody for glutamylation, GT335, which requires an acidic amino acid residue at the position following the glutamylation site (Wolff et al, 1992) (Fig 1A, yellow residues). This explains why GT335 had persistently shown very weak detection levels of β-tubulin in past studies and underpinned the need for a new detection tool. We thus raised polyclonal antibodies to a peptide mimicking the C-terminal tail of β2-tubulin (TubB2) with a single-glutamate branch at E435 (Fig 1B). This antibody was called β-monoE. Figure 1. TTLL1 and TTLL7 have distinct enzymatic specificities in brain and in neurons‡ A. Alignment of sequences of all human α- and β-tubulin isotypes. Only the C-terminal tail region of the tubulins is shown. Isotypes with identical amino acid sequence in these regions are pooled. A large number of glutamate residues in the C-terminal tails would, if modified by glutamylation, be detectable with GT335 (yellow), which requires one additional acidic amino acid residue after the modification site (Wolff et al, 1992). By contrast, the GEF motif (red/purple), which is the main glutamylation site in β2-tubulin (TUBB2) in the brain (Rüdiger et al, 1992), is present only in a few β-tubulin isotypes, but not in α-tubulin. B. Schematic representation of the synthetic peptide used as antigen to raise the β-monoE antibody. The modified glutamate residue is in red. C. Representative immunoblots of brain extracts of wild-type, Ttll1−/− and Ttll7−/− mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1−/− brain. By contrast, β-tubulin glutamylation, which is specifically detected with β-monoE, is absent from brains of Ttll7−/− mice. D. Representative immunoblots of extracts of wild-type, Ttll1−/− and Ttll7−/− primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1−/− neurons, while β-tubulin glutamylation lacks in Ttll7−/− neurons. E. Schematic representation of the epitopes detected by the anti-glutamylation antibodies used in this study. For clarity, α- and β-tubulin are shown separately. F, G. Immunocytochemistry of DIV4 primary hippocampal neurons from wild-type, Ttll1−/− and Ttll7−/− mice. Microtubules in Ttll1−/− cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), and Ttll7−/− neurons are specifically reduced in β-monoE reactivity, indicating the lack of β-tubulin glutamylation (G). Source data are available online for this figure. Source Data for Figure 1 [embj2021108498-sup-0004-SDataFig1.tif] Download figure Download PowerPoint To detect the levels of glutamylation on α- and β-tubulin, brain extracts from wild-type, Ttll1−/− and Ttll7−/− mice were separated on SDS–PAGE gels that allow a clear separation of α- and β-tubulin (Magiera & Janke, 2013). Additionally, we adjusted all samples to equal tubulin content in order to specifically highlight changes in levels and distribution of the tubulin PTMs. In wild-type brain, the novel β-monoE antibody detected a strong and specific signal on β-tubulin, while entirely omitting α-tubulin. By contrast, GT335 and polyE strongly labelled α-tubulin, but showed virtually no signal on β-tubulin (Fig 1C; wild type). This shows that, as predicted, the novel β-monoE antibody is specific to the glutamylated GEF motif found on β2-tubulin, but appears to not detect any other glutamylation site, given the complete lack of α-tubulin detection (Fig 1E). In the Ttll1−/− brains, the strong labelling of α-tubulin with GT335 and polyE was almost entirely lost, while the β-monoE antibody detected similar β-tubulin glutamylation levels as in wild-type brains (Fig 1C; Ttll1−/−). In Ttll7−/− brains, by contrast, only the β-monoE labelling disappeared, while GT335 and polyE remained unchanged (Fig 1C; Ttll7−/−). These observations demonstrate that in the nervous system, the glutamylation of α- and β-tubulin is independently controlled by TTLL1 and TTLL7, respectively. It also shows that—at least in the mature brain—TTLL1 and TTLL7 are the predominant polyglutamylating enzymes of tubulin. Importantly, our experiments also reveal that the loss of glutamylation on α- and β-tubulin in Ttll1−/− and Ttll7−/− brains is not compensated for by other TTLL enzymes. The independent and distinct roles of TTLL1 and TTLL7 in tubulin glutamylation are key prerequisites allowing these enzymes to independently control yet-to-be defined functions of the microtubule cytoskeleton. So far, we had characterised α- and β-tubulin in whole-brain extracts, which contain neurons as well as glia cells. To ascertain that both TTLL1 and TTLL7 are involved in the glutamylation of tubulin in neurons, we prepared primary neurons from Ttll1−/− and Ttll7−/− hippocampi and analysed them by immunoblot after 4 days of differentiation (days in vitro 4, DIV4). Similar to what we observed with the brain extracts (Fig 1C), Ttll1−/− neurons almost entirely lost the polyE signal on α-tubulin, while Ttll7−/− neurons selectively lacked β-tubulin glutamylation as revealed with β-monoE antibody (Fig 1D). This was confirmed by immunocytochemistry of primary hippocampal neurons: while polyE, but not β-monoE, labelling was decreased in Ttll1−/− cells (Fig 1F), a specific loss of β-monoE signal was observed in Ttll7−/− neurons, while the polyE signal remained unchanged (Fig 1G). Besides confirming that both TTLL1 and TTLL7 specifically modify α- and β-tubulin in neurons, this result also demonstrated that due to the specificity of these two antibodies (Fig 1C–E), they can be used to selectively detect α- and β-tubulin glutamylation by immunocytochemistry, which was so far not possible. In summary, TTLL1 and TTLL7 are the two key enzymes that catalyse polyglutamylation in the nervous system, and both enzymes are active in neurons. Under physiological conditions, they show strictly distinct activities: TTLL1 modifies α-tubulin, while TTLL7 modifies β-tubulin, and they do not compensate for each other if one of the two enzymes is inactivated. We thus hypothesised that both enzymes control neuronal functions in an independent manner. Distinct roles of TTLL1 and TTLL7 in Purkinje-cell degeneration We have previously demonstrated that an abnormal accumulation of polyglutamylation on both α- and β-tubulin leads to degeneration of various neuronal populations (Magiera et al, 2018). In some brain regions, such as the cerebellum, inactivation of a single deglutamylase, CCP1, was sufficient to induce hyperglutamylation and neurodegeneration. The most striking phenotype of Ccp1−/− mice is the degeneration of the cerebellar Purkinje cells within the first month of life (Mullen et al, 1976). We and others previously demonstrated that this degeneration can be prevented by the concomitant depletion or deletion of the glutamylase TTLL1 (Rogowski et al, 2010; Berezniuk et al, 2012). Moreover, we showed that the protective effect of TTLL1 loss is a cell-autonomous mechanism, as deleting TTLL1 selectively in Purkinje cells of Ccp1−/− mice (Ccp1−/−Ttll1flox/flox – cre-L7) fully prevented their degeneration for up to 20 months (Magiera et al, 2018). While these experiments have provided a definitive proof that accumulation of polyglutamylation generated by TTLL1 is the primary cause of the degeneration of Purkinje cells in Ccp1−/− mice, a direct link with the enzymatic specificity of TTLL1 has not been established. Having demonstrated that TTLL1 and TTLL7 are the two key enzymes involved in tubulin polyglutamylation in neurons, with each of them catalysing an exclusive subtype of glutamylation (Fig 1), we tested their individual involvements in the degeneration of Purkinje cells in mice lacking CCP1. For this, we generated two combinatorial mouse strains: Ccp1−/−Ttll1−/− and Ccp1−/−Ttll7−/−. As expected, Purkinje cells of Ccp1−/−Ttll1−/− mice were protected from degeneration. By contrast, this was not the case in Ccp1−/−Ttll7−/− mice, in which Purkinje cells, like in the Ccp1−/− mice, are entirely lost (Fig 2A). Moreover, degeneration of Purkinje cells in both Ccp1−/− and Ccp1−/−Ttll7−/− mice appears to follow a similar pattern of degeneration as seen at 25 days after birth, where we observed early signs of degeneration such as partial loss of Purkinje cells and axonal swellings specifically in Ccp1−/− and Ccp1−/−Ttll7−/− mice (Fig 2B). This demonstrates that TTLL1-catalysed polyglutamylation alone is sufficient to cause the neurodegeneration observed in Ccp1−/− mice, while TTLL7 appears to play no role in this process. Figure 2. TTLL1 plays a key role in Purkinje-cell degeneration and tubulin hyperglutamylation A. Representative histology images of cerebella from 3-month-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1−/−, Ttll7−/− and Ccp1−/−Ttll1−/− mice, in contrast to the complete absence of these cells in Ccp1−/− and Ccp1−/−Ttll7−/− mice. A complete representation of all analysed cerebella is shown in Fig EV1. B. Histology of 25-day-old mice with partial degeneration of Purkinje Cells. Note that both Ccp1−/− and Ccp1−/−Ttll7−/− brains show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; and orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration). C. Representative immunoblots of cerebellum extracts of wild-type, Ccp1−/−, Ttll7−/−, Ccp1−/−Ttll7−/−, Ttll1−/− and Ccp1−/−Ttll1−/− mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 leads to a loss of glutamylation from β-tubulin (β-monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE). Source data are available online for this figure. Source Data for Figure 2 [embj2021108498-sup-0005-SDataFig2.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Cerebella from all mice analysed in this study (complement to Fig 2A)Histology of cerebella from 3-month-old mice stained with anti-calbindin antibody. Note the presence of an intact Purkinje-cell layer in all cerebella of wild-type, Ttll1−/−, Ttll7−/− and Ccp1−/−Ttll1−/− mice, in contrast to the complete absence of these cells in Ccp1−/− and Ccp1−/−Ttll7−/− mice (*heterozygous alleles were present in some mice analysed here: Ccp1+/− (white*); Ttll1+/− (blue*); and Ttll7+/− (brown*)). Source data are available online for this figure. Download figure Download PowerPoint To understand the changes in polyglutamylation patterns underlying the different impacts of the two glutamylases on neurodegeneration, we analysed cerebella of the different mouse models by immunoblot with our set of glutamylation-specific antibodies (Fig 1E). The characteristic features of Ccp1−/− mice are a massive accumulation of polyE-reactive polyglutamylation, and a strong GT335 signal on β-tubulin, which is not seen in wild type (Fig 2C; Ccp1−/−). In Ccp1−/−Ttll7−/− brains, the β-monoE reactivity was completely lost, and GT335 also failed to detect the β-tubulin band. However, the massive polyE-reactive hyperglutamylation on α-tubulin persisted in the absence of TTLL7 (Fig 2C; Ccp1−/−Ttll7−/−). This was contrasted by a complete loss of polyE- and GT335-reactive hyperglutamylation on both α- and β-tubulin in Ccp1−/− mice lacking TTLL1 (Fig 2C; Ccp1−/−Ttll1−/−) that had been observed previously (Berezniuk et al, 2012). By contrast, the glutamylation of β-tubulin at E435, detected with β-monoE antibody, was similar to wild-type levels in the cerebella of Ccp1−/−Ttll1−/− mice, underpinning the unique role of TTLL7 in the modification of this site. These results imply that TTLL1 is the sole glutamylase involved in the generation of the abnormally high levels of polyglutamylation observed in mice lacking CCP1, while TTLL7 appears to play no critical role in the generation of hyperglutamylation. Nevertheless, we observed an accumulation of GT335-reactive β-tubulin glutamylation in mice lacking CCP1 (Fig 2C; Ccp1−/−), which initially suggested that TTLL7 could still be involved. However, even the abnormal increase in β-tubulin glutamylation appears to be dependent on TTLL1, as it was not seen in the Ccp1−/−Ttll1−/− mice (Fig 2C; Ccp1−/−Ttll1−/−). By demonstrating that TTLL7 alone does not lead to hyperglutamylation in the absence of CCP1, our observations provide the mechanistic background for the dominant role of TTLL1 in the generation of hyperglutamylation that leads to neurodegeneration of the Purkinje cells. Synergy of TTLL7 and TTLL1 in modifying different sites on β-tubulin Analyses using the new β-monoE antibody revealed that TTLL7 predominantly modifies E435 on β-tubulin, a glutamylation site that cannot be detected with GT335 (Fig 1E). In brain tissue of Ccp1−/− mice, however, β-tubulin is strongly labelled with GT335 (Fig 2C; Ccp1−/−), indicating that other sites are glutamylated (Fig 1A, yellow residues). Strikingly
Referência(s)