Francesca Maltecca - Academia.edu (original) (raw)
Papers by Francesca Maltecca
Brain
AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial me... more AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane (IMM). Heterozygous AFG3L2 mutations cause Spinocerebellar Ataxia type 28 (SCA28) or Dominant Optic Atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe Spastic Ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia, and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the over-activation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the IMM protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response (ISR). In general, the ISR reduces global protein synthesis and driv...
Oxidative Medicine and Cellular Longevity, Oct 31, 2019
Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more... more Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.
The Journal of Neuroscience, Mar 12, 2008
The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form... more The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form a supracomplex in charge of the essential protein quality control within mitochondria. Mutations of paraplegin cause a specific axonal degeneration of the upper motoneuron and, therefore, hereditary spastic paraplegia. Here we present two Afg3l2 murine models: a newly developed null and a spontaneous mutant that we found carrier of a missense mutation. Contrasting with the mild and late onset axonal degeneration of paraplegin-deficient mouse, Afg3l2 models display a marked impairment of axonal development with delayed myelination and poor axonal radial growth leading to lethality at P16. The increased severity of the Afg3l2 mutants is explained by two main molecular features that differentiate AFG3L2 from paraplegin: its higher neuronal expression and its versatile ability to support both hetero-oligomerization and homo-oligomerization. Our data assign to AFG3L2 a crucial role by linking mitochondrial metabolism and axonal development. Moreover, we propose AFG3L2 as an excellent candidate for motoneuron and cerebellar diseases with early onset unknown etiology.
Methods in molecular biology, 2010
Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major ta... more Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major target of oxidative damage. Defects in the mitochondrial respiratory chain complexes can increase ROS production and reduce ROS removal, leading to oxidative modification of proteins, lipids, and DNA. AAA proteases of the inner mitochondrial membrane, paraplegin and AFG3L2, participate in the biogenesis and maintenance of respiratory chain complexes. These proteins form hetero-oligomeric paraplegin/AFG3L2 and homo-oligomeric AFG3L2 complexes named m-AAA proteases. Inactivation of m-AAA proteases causes respiratory defects and altered mitochondrial morphology both in yeast and in mammals. In fact, mouse models defective for Afg3l2 display a very severe neurological syndrome and die within two weeks after birth. They display widespread morphological alterations of mitochondria in the central and peripheral nervous system and deficiencies in respiratory chain complex I and in complex III, which are major producers of ROS in physiological and especially in pathological conditions. Therefore, an efficient and reliable methodology to monitor the effect of increased ROS production is useful for accurately phenotyping cellular and animal models mutants in m-AAA. By measuring carbonyl formation as marker of protein oxidation, we have shown that respiratory defects cause oxidative damage in Afg3l2 mutants, indicating that oxidative stress is crucial in the pathogenesis of m-AAA deficiency.
Molecular Autism
Background Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of... more Background Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population. Methods We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality. Results Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons...
Journal of World Mitochondria Society, 2015
In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse ... more In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse model of spinocerebellar ataxia 28 (SCA28) characterized by dark degeneration of Purkinje cells. Primary cortical neurons from mice, WT and KO for Afg3l2 (the mitochondrial gene mutated in SCA28), were analyzed by single-cell videomicroscopy after iron overload, a condition able to to promote oxidative stress. Different fluorescent probes were employed to monitor iron oxidative status, ROS formation, labile iron pool, glutathione content and cell death. Detoxifying proteins were quantified by Western blot. Upon iron overload, Afg3l2-KO cortical neurons showed a significantly higher percentage of cell death than the WT counterpart. Surprisingly, with culture time, Afg3l2-KO neurons acquired a protective phenotype that made them more resistant than WT neurons to oxidative conditions. Further investigation revealed that the main antioxidant cellular molecule, glutathione, was not involved i...
Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense ... more Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significan...
Oxidative Medicine and Cellular Longevity, 2019
Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more... more Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the ...
Journal of Cell Science, 2018
The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 (... more The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 (i-AAA protease complex) and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 (m-AAA complex) as the major protease mediating this event by maturing the pre-pro-OMA1 of 60 kDa to the pro-OMA1 form of 40 kDa by severing the amino-terminal part without recognizing specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently OPA1 isoforms, thus adding new information in the comprehension of the molecular mechanisms in mitochondrial dynamics and of neurodegenerative diseases affected by these phenomena.
BMC Medical Genomics, 2013
Background SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We perfor... more Background SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. Methods Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. Results We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an incre...
JCI insight, Jun 22, 2023
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca 2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca 2+-evoked responses in Sacs-/-PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca 2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/cerebellum. Based on this pathogenetic cascade, we treated Sacs-/mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca 2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/mice, at both pre-and post-symptomatic stages. We correlated this effect to restored Ca 2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACS patients.
JCI insight, Jun 22, 2023
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca 2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca 2+-evoked responses in Sacs-/-PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca 2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/cerebellum. Based on this pathogenetic cascade, we treated Sacs-/mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca 2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/mice, at both pre-and post-symptomatic stages. We correlated this effect to restored Ca 2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACS patients.
Neurology, 2021
Background and ObjectivesAutosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is ca... more Background and ObjectivesAutosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level.MethodsWe analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein.ResultsWe found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in pati...
Human Molecular Genetics, 2019
Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological st... more Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5′-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense ...
Human molecular genetics, May 23, 2017
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the ... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments.To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations.Reorganisation of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumu...
Acta Neuropathologica Communications, 2020
Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilatera... more Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type ...
Autosomal recessive spastic ataxia of Charlevoix-Saguenay is caused by more than 200 different mu... more Autosomal recessive spastic ataxia of Charlevoix-Saguenay is caused by more than 200 different mutations in the SACS gene encoding sacsin, a huge multimodular protein of unknown function. ARSACS phenotypic spectrum is highly variable. Previous studies correlated the nature and position of SACS mutations with age of onset or disease severity, though the effects on protein stability were not considered.In this study, we explain mechanistically the lack of genotype-phenotype correlation in ARSACS, with important consequences for disease diagnosis and treatment.We found that sacsin is almost absent in ARSACS fibroblasts, regardless of the nature of the mutation. We did not detect sacsin in patients with truncating mutations, while we found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense mutations. We excluded SACS mRNA decay, defective translation, or faster post-translational degradation as causes of protein reduction. Conversely, we demonstrate...
Brain
AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial me... more AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane (IMM). Heterozygous AFG3L2 mutations cause Spinocerebellar Ataxia type 28 (SCA28) or Dominant Optic Atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe Spastic Ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia, and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the over-activation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the IMM protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response (ISR). In general, the ISR reduces global protein synthesis and driv...
Oxidative Medicine and Cellular Longevity, Oct 31, 2019
Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more... more Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.
The Journal of Neuroscience, Mar 12, 2008
The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form... more The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form a supracomplex in charge of the essential protein quality control within mitochondria. Mutations of paraplegin cause a specific axonal degeneration of the upper motoneuron and, therefore, hereditary spastic paraplegia. Here we present two Afg3l2 murine models: a newly developed null and a spontaneous mutant that we found carrier of a missense mutation. Contrasting with the mild and late onset axonal degeneration of paraplegin-deficient mouse, Afg3l2 models display a marked impairment of axonal development with delayed myelination and poor axonal radial growth leading to lethality at P16. The increased severity of the Afg3l2 mutants is explained by two main molecular features that differentiate AFG3L2 from paraplegin: its higher neuronal expression and its versatile ability to support both hetero-oligomerization and homo-oligomerization. Our data assign to AFG3L2 a crucial role by linking mitochondrial metabolism and axonal development. Moreover, we propose AFG3L2 as an excellent candidate for motoneuron and cerebellar diseases with early onset unknown etiology.
Methods in molecular biology, 2010
Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major ta... more Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major target of oxidative damage. Defects in the mitochondrial respiratory chain complexes can increase ROS production and reduce ROS removal, leading to oxidative modification of proteins, lipids, and DNA. AAA proteases of the inner mitochondrial membrane, paraplegin and AFG3L2, participate in the biogenesis and maintenance of respiratory chain complexes. These proteins form hetero-oligomeric paraplegin/AFG3L2 and homo-oligomeric AFG3L2 complexes named m-AAA proteases. Inactivation of m-AAA proteases causes respiratory defects and altered mitochondrial morphology both in yeast and in mammals. In fact, mouse models defective for Afg3l2 display a very severe neurological syndrome and die within two weeks after birth. They display widespread morphological alterations of mitochondria in the central and peripheral nervous system and deficiencies in respiratory chain complex I and in complex III, which are major producers of ROS in physiological and especially in pathological conditions. Therefore, an efficient and reliable methodology to monitor the effect of increased ROS production is useful for accurately phenotyping cellular and animal models mutants in m-AAA. By measuring carbonyl formation as marker of protein oxidation, we have shown that respiratory defects cause oxidative damage in Afg3l2 mutants, indicating that oxidative stress is crucial in the pathogenesis of m-AAA deficiency.
Molecular Autism
Background Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of... more Background Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population. Methods We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality. Results Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons...
Journal of World Mitochondria Society, 2015
In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse ... more In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse model of spinocerebellar ataxia 28 (SCA28) characterized by dark degeneration of Purkinje cells. Primary cortical neurons from mice, WT and KO for Afg3l2 (the mitochondrial gene mutated in SCA28), were analyzed by single-cell videomicroscopy after iron overload, a condition able to to promote oxidative stress. Different fluorescent probes were employed to monitor iron oxidative status, ROS formation, labile iron pool, glutathione content and cell death. Detoxifying proteins were quantified by Western blot. Upon iron overload, Afg3l2-KO cortical neurons showed a significantly higher percentage of cell death than the WT counterpart. Surprisingly, with culture time, Afg3l2-KO neurons acquired a protective phenotype that made them more resistant than WT neurons to oxidative conditions. Further investigation revealed that the main antioxidant cellular molecule, glutathione, was not involved i...
Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense ... more Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significan...
Oxidative Medicine and Cellular Longevity, 2019
Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more... more Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the ...
Journal of Cell Science, 2018
The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 (... more The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 (i-AAA protease complex) and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 (m-AAA complex) as the major protease mediating this event by maturing the pre-pro-OMA1 of 60 kDa to the pro-OMA1 form of 40 kDa by severing the amino-terminal part without recognizing specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently OPA1 isoforms, thus adding new information in the comprehension of the molecular mechanisms in mitochondrial dynamics and of neurodegenerative diseases affected by these phenomena.
BMC Medical Genomics, 2013
Background SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We perfor... more Background SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. Methods Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. Results We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an incre...
JCI insight, Jun 22, 2023
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca 2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca 2+-evoked responses in Sacs-/-PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca 2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/cerebellum. Based on this pathogenetic cascade, we treated Sacs-/mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca 2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/mice, at both pre-and post-symptomatic stages. We correlated this effect to restored Ca 2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACS patients.
JCI insight, Jun 22, 2023
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca 2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca 2+-evoked responses in Sacs-/-PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca 2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/cerebellum. Based on this pathogenetic cascade, we treated Sacs-/mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca 2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/mice, at both pre-and post-symptomatic stages. We correlated this effect to restored Ca 2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACS patients.
Neurology, 2021
Background and ObjectivesAutosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is ca... more Background and ObjectivesAutosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level.MethodsWe analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein.ResultsWe found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in pati...
Human Molecular Genetics, 2019
Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological st... more Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5′-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense ...
Human molecular genetics, May 23, 2017
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the ... more Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments.To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations.Reorganisation of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumu...
Acta Neuropathologica Communications, 2020
Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilatera... more Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type ...
Autosomal recessive spastic ataxia of Charlevoix-Saguenay is caused by more than 200 different mu... more Autosomal recessive spastic ataxia of Charlevoix-Saguenay is caused by more than 200 different mutations in the SACS gene encoding sacsin, a huge multimodular protein of unknown function. ARSACS phenotypic spectrum is highly variable. Previous studies correlated the nature and position of SACS mutations with age of onset or disease severity, though the effects on protein stability were not considered.In this study, we explain mechanistically the lack of genotype-phenotype correlation in ARSACS, with important consequences for disease diagnosis and treatment.We found that sacsin is almost absent in ARSACS fibroblasts, regardless of the nature of the mutation. We did not detect sacsin in patients with truncating mutations, while we found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense mutations. We excluded SACS mRNA decay, defective translation, or faster post-translational degradation as causes of protein reduction. Conversely, we demonstrate...