Francesca Maltecca

Intergenerational instability and marked anticipation in SCA-17

The authors describe an Italian family with autosomal dominant ataxia, dementia, psychiatric and extrapyramidal features, epilepsy, mild sensorimotor axonal neuropathy, and MRI findings of cerebral and cerebellar atrophy. A child had a distinctive presentation with onset at 3 years, growth retardation, fast progression, and early death. Molecular analysis demonstrated an expanded CAG/CAA repeat in the TBP gene (SCA-17). The repeat size was 66 triplets in the child and 53 in all the other patients.

Haploinsufficiency of AFG3L2, the gene responsible for spinocerebellar ataxia type 28, causes mitochondria-mediated Purkinje cell dark degeneration.

Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-oligomeric paraplegin-AFG3L2 and homo-oligomeric AFG3L2 complexes in the inner mitochondrial membrane, named m-AAA proteases. These complexes ensure protein quality control in the inner membrane, jointly with a chaperone-like activity on the respiratory chain complexes. Despite coassembling in the same complex, mutations of either paraplegin or AFG3L2 cause two different neurodegenerative disorders. Indeed, mutations of paraplegin are responsible for a recessive form of hereditary spastic paraplegia, whereas mutations of AFG3L2 have been recently associated to a dominant form of spinocerebellar ataxia (SCA28). In this work, we report that the mouse model haploinsufficient for Afg3l2 recapitulates important pathophysiological features of the human disease, thus representing the first SCA28 model. Furthermore, we propose a pathogenetic mechanism in which respiratory chain dysfunction and increased reactive oxygen species production caused by Afg3l2 haploinsufficiency lead to dark degeneration of Purkinje cells and cerebellar dysfunction.

The Mitochondrial Protease AFG3L2 Is Essential for Axonal Development

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.

Dementia, ataxia, extrapyramidal features, and epilepsy: phenotype spectrum in two Italian families with spinocerebellar ataxia type 17.

Abstract.We observed two families with a dominantly inherited complex neurological syndrome with onset in adulthood. Family F included 9 affected in four generations. One patient showed prominent anticipation of onset age. Onset was with cerebellar signs followed by dementia, psychiatric symptoms, seizures, and extrapyramidal features. Family M included 14 affected individuals in five generations. Presenting symptoms were either psychiatric and cognitive impairment or a cerebellar syndrome. Extrapyramidal features, dysphagia, incontinence, seizures, and myoclonus may occur. In both families magnetic resonance imaging showed marked atrophy of the brain and cerebellum. Molecular analyses demonstrated an expanded CAG/CAA repeat in the in the TATA box-binding protein (TBP) gene (SCA17).

Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca2+ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca2+ homeostasis nor of the reduced driving force for Ca2+ internalization within mitochondria, since cytosolic Ca2+ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca2+ uptake speed in Afg3l2−/− cells, indicating the presence of functional Ca2+ uptake machinery. Our results show that the defective Ca2+ handling in Afg3l2−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca2+ elevations, hampering the proper Ca2+ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca2+ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca2+ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.

Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice

Cells rely on efficient protein quality control systems (PQCs) to maintain proper activity of mitochondrial proteins. As part of this system, the mitochondrial chaperone Hsp60 assists folding of matrix proteins and it is an essential protein in all organisms. Mutations in Hspd1, the gene encoding Hsp60, are associated with two human inherited diseases of the nervous system, a dominantly inherited form of spastic paraplegia (SPG13) and an autosomal recessively inherited white matter disorder termed MitCHAP60 disease. Although the connection between mitochondrial failure and neurodegeneration is well known in many neurodegenerative disorders, such as Huntingtons disease, Parkinsons disease, and hereditary spastic paraplegia, the molecular basis of the neurodegeneration associated with these diseases is still ill-defined. Here, we investigate mice heterozygous for a knockout allele of the Hspd1 gene encoding Hsp60. Our results demonstrate that Hspd1 haploinsufficiency is sufficient to cause a late onset and slowly progressive deficit in motor functions in mice. We furthermore emphasize the crucial role of the Hsp60 chaperone in mitochondrial function by showing that the motor phenotype is associated with morphological changes of mitochondria, deficient ATP synthesis, and in particular, a defect in the assembly of the respiratory chain complex III in neuronal tissues. In the current study, we propose that our heterozygous Hsp60 mouse model is a valuable model system for the investigation of the link between mitochondrial dysfunction and neurodegeneration.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BackgroundSCA28 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.MethodsGene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR.ResultsWe 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 increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways.ConclusionsWhole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

In Vivo Detection of Oxidized Proteins: A Practical Approach to Tissue-Derived Mitochondria

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.

m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics

ABSTRACT The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (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 [matrix (m)-AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena. This article has an associated First Person interview with the first author of the paper. Summary: AFG3L2 and YME1L1 coordinately regulate OMA1 maturation and consequently, mitochondrial dynamics.