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 Huntington's disease, Parkinson's 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.

Genetic interaction between the m-AAA protease isoenzymes reveals novel roles in cerebellar degeneration.

The mitochondrial m-AAA protease has a crucial role in axonal development and maintenance. Human mitochondria possess two m-AAA protease isoenzymes: a hetero-oligomeric complex, composed of paraplegin and AFG3L2 (Afg3 like 2), and a homo-oligomeric AFG3L2 complex. Loss of function of paraplegin (encoded by the SPG7 gene) causes hereditary spastic paraplegia, a disease characterized by retrograde degeneration of cortical motor axons. Spg7(-/-) mice show a late-onset degeneration of long spinal and peripheral axons with accumulation of abnormal mitochondria. In contrast, Afg3l2(Emv66/Emv66) mutant mice, lacking the AFG3L2 protein, are affected by a severe neuromuscular phenotype, due to defects in motor axon development. The role of the homo-oligomeric m-AAA protease and the extent of cooperation and redundancy between the two isoenzymes in adult neurons are still unclear. Here we report an early-onset severe neurological phenotype in Spg7(-/-) Afg3l2(Emv66/+) mice, characterized by loss of balance, tremor and ataxia. Spg7(-/-) Afg3l2(Emv66/+) mice display acceleration and worsening of the axonopathy observed in paraplegin-deficient mice. In addition, they show prominent cerebellar degeneration with loss of Purkinje cells and parallel fibers, and reactive astrogliosis. Mitochondria from affected tissues are prone to lose mt-DNA and have unstable respiratory complexes. At late stages, neurons contain structural abnormal mitochondria defective in COX-SDH reaction. Our data demonstrate genetic interaction between the m-AAA isoenzymes and suggest that different neuronal populations have variable thresholds of susceptibility to reduced levels of the m-AAA protease. Moreover, they implicate impaired mitochondrial proteolysis as a novel pathway in cerebellar degeneration.

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 heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase.

BACKGROUND: Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy. METHODS: We used a range of biological and biochemical in vitro assays to assess the effect of arimoclomol on GCase activity in ex vivo systems of primary fibroblasts and neuronal-like cells from GD patients. FINDINGS: We found that arimoclomol induced relevant HSPs such as ER-resident HSP70 (BiP) and enhanced the folding, maturation, activity, and correct cellular localization of mutated GCase across several genotypes including the common L444P and N370S mutations in primary cells from GD patients. These effects where recapitulated in a human neuronal model of GD obtained by differentiation of multipotent adult stem cells. INTERPRETATION: These data demonstrate the potential of HSP-targeting therapies in GCase-deficiencies and strongly support the clinical development of arimoclomol as a potential therapeutic option for the neuronopathic forms of GD. FUNDING: The research was funded by Orphazyme A/S, Copenhagen, Denmark.

Molecular chaperone disorders: defective Hsp60 in neurodegeneration.

Chaperonins, a subgroup of molecular chaperones, form ring-shaped structures and assist folding of proteins by enclosing them in their inner cavity. The mitochondrial Hsp60/Hsp10 chaperonin system is essential for cell viability and only a very small number of mutations causing human disease have so far been found that appear to selectively affect neuronal tissues. We here review the knowledge on the mammalian Hsp60/Hsp10 system and discuss evidence and observations, which may explain why this is the case. The Hsp60 mutations shown to be associated with neurodegenerative diseases mildly affect the protein and leave residual function. We present arguments for the notion that the neuron/glia specificity may be due to an effect of Hsp60 deficiency on myelination, a neuron-specific property. The substrates of the Hsp60/Hsp10 system are only poorly defined, but the combination of deficiency of a number of mitochondrial enzymes and proteins that are highly dependent on this system for folding is the likely trigger for deficient myelination. However, a number of experimental observations indicate that Hsp60 may also have roles outside mitochondria and deficiency of Hsp60 due to mutation may also affect myelination via these signaling pathways. Taken together, it appears that mild Hsp60 deficiency primarily affects neuronal and/or glia cells whereas more severe deficiency of Hsp60 would affect all tissues and not be compatible with life. We discuss in the end what approaches may lead to a further understanding of the functions of the Hsp60/Hsp10 system in mammalian cells and thus its role in disease conditions.