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GATM Cause a In kidneys, GATM expression is restricted to proximal tu- bular cells (PTCs), which house one of the largest mitochon- Dominant Fibrillar drial compartments in the human body to support the high Conformational Disease in energy demand for their titanic transport activity. This ex- plained the specific PTC damage related to GATM disease Mitochondria—When Eternity (thus RFS). Remarkably, GATM immunogold labeling of a pa- Kills tient kidney biopsy decorated fibrillary aggregates in mito- chondrial PTCs. To address the mechanism of fibril formation by a cell biologic approach, the authors generated stable trans- Pierre J. Courtoy and Patrick Henriet fectants in proximal tubular LLC-PK1 cells for tetracyclin- Cell Biology Unit, de Duve Institute, Université Catholique de inducible overexpression of wild-type human GATM and Louvain, Brussels, Belgium each of its four known point mutants. Wild-type GATM over- J Am Soc Nephrol 29: ccc–ccc, 2018. expression had no effect on mitochondria, but all mutants doi: https://doi.org/10.1681/ASN.2018040450 caused a dramatic structural mitochondrial deformation, re- sembling sickle erythrocytes. Such mitochondria reproduced the immunogold pattern of the patient biopsy. Transfectants More than 60 years ago, Luder and Sheldon1 and then, other elegantly served to show irreversibility of the deformation by nephrologists reported on a rare form of autosomal dominant aggregates on removal of the tetracyclin inducer, much beyond renal Fanconi syndrome (RFS) that appeared in childhood the normal mitochondrial turnover time. Longitudinal fibril- and slowly evolved toward kidney insufficiency. These clinical lary aggregation was found to prevent mitochondrial dynamics features were thus quite distinct from nephropathic cystinosis, (fission), trigger reactive oxygen species (ROS) production, and the most frequent cause of hereditary (recessive) RFS.2 Over activate the NLRP3 inflammasome. the years, additional families were described, and the affected The paper culminates in proposing an explanation for the locus was identified; however, the causative and disease propensity for aggregation by molecular dynamic modeling. mechanism remained elusive. In this issue of the Journal of the Normal GATM acts as a homodimer (A:A). All mutations were American Society of Nephrology, the paper by Reichold et al.3 predicted in silico to generate an additional pathogenic b-sheet– finally defines the mutated gene as GATM with identification interacting interface (A/A) supporting alternate bead-like elon- of another family with de novo , explains the dominant gation, thus converting normal dimers into longitudinal mode of transmission by an attractive multimerization-based multimers (A:A/A:A/A...; i.e., fibrillary aggregates). Incidentally, fibrillar molecular model, and further proposes a rational die- it is remarkable that, among the 1500 predicted mitochondrial tary treatment that remains to be tested. proteins, GATM mutations are so far unique in this behavior. GATM encodes for a nuclear-encoded mitochondrial matrix Abnormal b-sheet formation is well known to favor bead-like dimeric , glycine amidinotransferase (hence GATM; also fibrillar polymerization in a1-antitrypsin affected by homozy- known as L-arginine:glycine amidinotransferase), catalyzing the gous Z mutation (PIZZ) to cause liver cirrhosis and a variety of penultimate reaction of the biosynthesis pathway. brain neurodegenerative diseases causing dementia.4 In PIZZ Fortunately, much is known about GATM, including infor- hepatocytes, bead-like polymers and entangled polymeric aggre- mation from crystallographic studies. All patients with GATM gates accrue slowly over months/years. Interestingly, unequal disease exhibited a single heterozygous missense mutation at exposure to inflammatory episodes explains in part the variable evolutionary conserved proline or threonine residues clustered age of cirrhosis onset due to changes in the rate of PIZZ synthesis in a small central stretch representing ,5% of the protein. As in the acute-phase response. This indicates that fibrillary disease predicted from dominant transmission, disease-causing GATM evolution may be influenced by intervention on monomer syn- mutations did not abolish enzymatic activity. Furthermore, thesis. Dominant mutations of the microtubule-associated GATM haploinsufficient mice had no significant kidney t-protein induce neurofibrillary lesions, which cause frontotem- phenotype, opening the possibility of suppressive intervention. poral dementia and are identical to those prevalent in acquired Alzheimer disease. Most recently, atomic resolution of t-fibrils, – Published online ahead of print. Publication date available at www.jasn.org. achieved by cryoelectron microscopy, revealed a typical cross b-structure.5 Thus, the predicted propensity to aggregation Correspondence: Prof. Pierre J. Courtoy or Prof. Patrick Henriet, Cell Biology Unit (CELL), de Duve Institute, Université Catholique de Louvain, 75, Avenue being dominant readily explains the autosomal dominant Hippocrate, Box B1.75.05, B-1200 Brussels, Belgium. Email: pierre.courtoy@ transmission and the slow progression of GATM disease; it uclouvain.be or [email protected] places this disease in the family of fibrillar conformational Copyright © 2018 by the American Society of Nephrology diseases and suggests the possibility of beneficial intervention

J Am Soc Nephrol 29: ccc–ccc,2018 ISSN : 1046-6673/2907-ccc 1 EDITORIALS www.jasn.org by decreasing synthesis of the specific protein culprit. Valida- GATM disease frequency, which is especially justified if effec- tion of molecular modeling of GATM fibrils by the recently tive treatment can indeed be proposed. As is the case for other available high-resolution cryoelectron microscopy might be human in biosynthetic pathways downregulated by considered. the end product, this paper further shows that creatine supple- How do fibrils escape clearance to cause disease? Protein mentation to wild-type mice at equivalent acceptable doses for aggregation is a central issue in biology and disease. Appro- humans significantly decreases GATM expression and thus, priate formation of intra- and extracellular fibrils has been might slow down disease progression (analogous to the control remarkably tamed by evolution (e.g., to generate cystoskeletal of inflammatory status in PIZZ patients). Whether creatine and collagen fibers). In the cytosol, labile actin-based micro- would prevent fibril formation in the transgenic LLC-PK1 cells filaments and tubulin-based microtubules undergo permanent could not be tested, because the endogenous promotor was remodeling by assembly at the “plus” end and depolymeriza- swapped with the tetracycline-inducible promotor. A knock- tion at the “minus” end (unless stabilized by drugs, such as in animal model (e.g., by CRISPR/Cas9 technology) is eagerly taxol). Also, in the secretory apparatus, premature intracellular awaited to not only further investigate pathogeny in vivo (in collagen fibril formation is prevented by the addition of tran- particular, natural course and disease adaptations that might sient, extracellularly cleavable globular termini at the two ends open unexpected avenues)10 but also, test effectiveness of cre- of the newly synthesized triple helix. Unwanted aggregation of atine supplementation. cytosolic proteins into irreversible fibrils is normally prevented by chaperone proteins, such as heat-shock proteins, and aggregates are normally cleared by the proteasome, selective macroau- tophagy, or chaperone-mediated autophagy. In the endoplas- ACKNOWLEDGMENTS mic reticulum of PIZZ hepatocytes, fibrils preserve the folding of individual monomers, which prevents recognition by chap- The investigations of P.J.C. are supported by the Cystinosis Research erones and thus, evades proteolytic clearance.4 Nuclear- Foundation. encoded mitochondrial matrix proteins are first assembled by free cytosolic ribosomes and remain unfolded thanks to specific DISCLOSURES cytosolic chaperones until they cross the double mitochondrial membrane by unidirectional transport systems; only after that None. do they fold (and can dimerize). As in their bacterial ancestors, mitochondria contain folding chaperones (e.g., mtHSP70) and REFERENCES proteases competent to clear misfolded (e.g., aged) proteins.6 Alternatively, misfolded ubiquitinated mitochondrial matrix 1. Luder J, Sheldon W: A familial tubular absorption defect of glucose – proteins can be retrotranslocated into the cytosol for degrada- and amino acids. Arch Dis Child 30: 160 164, 1955 7 2. Cherqui S, Courtoy PJ: The renal Fanconi syndrome in cystinosis: tion by the proteasome. It can, however, be suspected that, like Pathogenic insights and therapeutic perspectives. Nat Rev Nephrol in PIZZ proteins, preservation of mutated GATM monomer 13: 115–131, 2017 structure in bead-like polymers prevents their recognition by 3. Reichold M, Klootwijk ED, Reinders J, Otto EA, Milani M, Broeker C, chaperone and proteolytic clearance, allowing for fibril exten- et al.: Glycine amidinotransferase (GATM), renal Fanconi syn- sion and aggregation. Furthermore, large mitochondrial fibrils drome, and kidney failure [published online ahead of print April 13, 2018]. J Am Soc Nephrol doi: 10.1681/ASN.2017111179 should obviously not be eligible for retrotranslocation into the 4. Carrell RW, Lomas DA: Alpha1-antitrypsin deficiency–a model for cytosol. As a final quality control, mitochondria constantly conformational diseases. N Engl J Med 346: 45–53, 2002 rejuvenate by fission/fusion,8 and defective mitochondria are 5. Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer disposed of by mitophagy. However, when GATM mutant mi- HJ, et al.: Cryo-EM structures of tau filaments from Alzheimer’s – tochondria are rigidified by transversal fibril bundles, fission is disease. Nature 547: 185 190, 2017 “ ” 6. Ryan MT, Naylor DJ, Høj PB, Clark MS, Hoogenraad NJ: The role of prevented. By analogy to frustrated phagocytosis, which occurs molecular chaperones in mitochondrial protein import and folding. Int 9 when extracellular preys are too large to be engulfed, huge Rev Cytol 174: 127–193, 1997 mitochondria deformed by longitudinal GATM fibers could 7. Bragoszewski P, Turek M, Chacinska A: Control of mitochondrial bio- predictably no longer be wrapped by the autophagosome mem- genesis and function by the ubiquitin-proteasome system. Open Biol 7: brane, causing “frustrated autophagy.” Consequences easily fol- 170007, 2017 8. Wai T, Langer T: Mitochondrial dynamics and metabolic regulation. low. Healthy mitochondria keep ROS production under control Trends Endocrinol Metab 27: 105–117, 2016 by their complex electron transfer system. Conversely, aging mi- 9. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, tochondria release abundant ROS, which trigger the inflamma- et al.: Multi-walled carbon nanotube induced frustrated phagocytosis, some as documented here. cytotoxicity and pro-inflammatory conditions in macrophages are What are new perspectives? The paper by Reichold et al.3 length dependent and greater than that of asbestos. Toxicol In Vitro 29: 1513–1528, 2015 stresses how important in-depth genetic analysis of unex- 10. Gaide Chevronnay HP, Janssens V, Van Der Smissen P, N’Kuli F, Nevo N, plained causes of RFS is, including identification of de novo Guiot Y, et al.: Time course of pathogenic and adaptation mechanisms in mutations. This will allow, in particular, a better estimate of cystinotic mouse kidneys. J Am Soc Nephrol 25: 1256–1269, 2014

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