SCA28 is one of the 31 known subtypes of autosomal dominant Spinocerebellar Ataxias (SCA) and it is caused by mutations in the AFG3L2 gene, encoding for an ATP-dependent metalloprotease belonging to the AAA-superfamily (ATPases Associated with a variety of cellular Activities). AFG3L2 can form homo- or hetero-oligomeric complexes with paraplegin: both reside in the Inner Mitochondrial Membrane (IMM) and exert protein quality surveillance and mediate protein processing. SCA28 patients mainly have missense mutations in the peptidase domain of AFG3L2. Two Belgian families were described with a partial deletion of exons 14 to 16, but the function altered by the mutations is still unclear. To further evaluate the role of missense changes, we generated a knockin (KI) mouse model carrying the p.M665R mutation (human p.M666R, showing the earliest onset). Homozygous Afg3l2M665R/M665R mice were lethal perinatally. Mice heterozygous for a SCA28 mutation (Afg3l2M665R/+) at 18 months of age showed a significant motor impairment in the beam balance test. However, in the repeated rotarod test, which detects motor learning deficits, their performance was indistinguishable from that of wild-type control littermates, suggesting a very mild cerebellar impairment. In all cerebellar lobules the number and the dendritic structure of Purkinje cells, and the thickness of the molecular and granular layers were almost identical in Afg3l2M665R/+ and wild-type mice. We studied mitochondrial dynamics in Mouse Embryonic Fibroblasts (MEFs) from KI and WT animals: (i) AFG3L2 protein levels were comparable; (ii) OPA1, a protein found in the IMM, involved in mitochondria fusion process, showed an increase of the short forms and absence of the long forms; (iii) mitochondrial network morphology by mitoRED staining suggested a complete fragmented pathway in homozygous KI and an intermediate tubular/fragmented network in heterozygous MEFs. No others fission/fusion proteins was shown deregulated. Taken together, these data show an impairment of OPA1 processing that results in increased mitochondrial fragmentation. This is in accordance with the cellular phenotype seen in Afg3l2 knockout mouse model, corroborating the idea that SCA28 mutations hitting the peptidase domain negatively impact on m-AAA complex function, probably acting as hypomorphic.
Spinocerebellar ataxia type 28: cellular and animal models to unravel the pathogenesis and to identify potential therapeutic targets
MANCINI, CECILIA;HOXHA, ERIOLA;TURCO, Emilia;ALTRUDA, Fiorella;TEMPIA, Filippo;BRUSCO, Alfredo
2015-01-01
Abstract
SCA28 is one of the 31 known subtypes of autosomal dominant Spinocerebellar Ataxias (SCA) and it is caused by mutations in the AFG3L2 gene, encoding for an ATP-dependent metalloprotease belonging to the AAA-superfamily (ATPases Associated with a variety of cellular Activities). AFG3L2 can form homo- or hetero-oligomeric complexes with paraplegin: both reside in the Inner Mitochondrial Membrane (IMM) and exert protein quality surveillance and mediate protein processing. SCA28 patients mainly have missense mutations in the peptidase domain of AFG3L2. Two Belgian families were described with a partial deletion of exons 14 to 16, but the function altered by the mutations is still unclear. To further evaluate the role of missense changes, we generated a knockin (KI) mouse model carrying the p.M665R mutation (human p.M666R, showing the earliest onset). Homozygous Afg3l2M665R/M665R mice were lethal perinatally. Mice heterozygous for a SCA28 mutation (Afg3l2M665R/+) at 18 months of age showed a significant motor impairment in the beam balance test. However, in the repeated rotarod test, which detects motor learning deficits, their performance was indistinguishable from that of wild-type control littermates, suggesting a very mild cerebellar impairment. In all cerebellar lobules the number and the dendritic structure of Purkinje cells, and the thickness of the molecular and granular layers were almost identical in Afg3l2M665R/+ and wild-type mice. We studied mitochondrial dynamics in Mouse Embryonic Fibroblasts (MEFs) from KI and WT animals: (i) AFG3L2 protein levels were comparable; (ii) OPA1, a protein found in the IMM, involved in mitochondria fusion process, showed an increase of the short forms and absence of the long forms; (iii) mitochondrial network morphology by mitoRED staining suggested a complete fragmented pathway in homozygous KI and an intermediate tubular/fragmented network in heterozygous MEFs. No others fission/fusion proteins was shown deregulated. Taken together, these data show an impairment of OPA1 processing that results in increased mitochondrial fragmentation. This is in accordance with the cellular phenotype seen in Afg3l2 knockout mouse model, corroborating the idea that SCA28 mutations hitting the peptidase domain negatively impact on m-AAA complex function, probably acting as hypomorphic.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
