Hsp60 Deficiency and Neurodegeneration; Investigations in Cellular and Mouse Models

Hsp60 deficiency and neurodegeneration; investigations in cellular and mouse models.

Peter Bross1, Raffaella Magnoni1, Anne Bie1, Johan Palmfeldt1, Jane. H. Christensen2, Mark West3, Ernst-Martin Füchtbauer4, Jakob Hansen1, Niels Gregersen1, Thomas J. Corydon2

Mutations in the HSPD1 gene encoding the human mitochondrial Hsp60 chaperone are associated with an autosomal dominant form of hereditary spastic paraplegia and a recessively inherited fatal hypomyelinating leukodystrophy (1-3). Based on our studies on the effects of the disease-associated Hsp60 mutations (1,4), we suggest that the residual levels of Hsp60 activity correlate to disease severity and age of onset. To test this hypothesis and to characterize the molecular disease pathogenesis we have produced a series of cellular models and a mouse model for Hsp60 deficiency.

We have generated a mutant mouse line bearing an inactivating genetrap insertion in the murine Hspd1 gene. Homozygous Hspd1(-/-) embryos died shortly after implantation demonstrating that Hsp60 is essential for early embryonic development (5). Heterozygous Hspd1(+/-) mice were born at the expected ratio and displayed significantly decreased levels of the Hspd1 transcript and concomitantly decreased amounts of Hsp60 protein in all tissues examined. Hsp60(+/-) mice back-crossed to C57/Bl6 background have a normal survival rate and development, however, they show motor impairment and signs of neurodegeneration. Motor impairment becomes evident at 12 months of age using the rotarod test. This is associated with abnormal extension reflex of the hindlimbs and a clasping tendency. The effects of decreased Hsp60 levels on mitochondrial protein composition in cortex and spinal cord is currently investigated by advanced mass spectrometry and first experiments show decreased levels of a series of mitochondrial proteins. These proteins may be involved in the pathogenesis.

With the Flp-In T-Rex system (InVitrogen) and human HEK293 cells we have generated mammalian cell lines allowing inducible expression of Hsp60 variant proteins carrying an artificially constructed dominant negative mutation and one carrying the Val98Ile variation associated spastic paraplegia SPG13. Upon induction of the integrated gene variants 2-3-fold overexpression is observed. Previous studies have indicated that these mutant variants will, together with the endogenous wild type Hsp60, be assembled into heterologous chaperone complexes. These cell lines are currently used to delineate the effects of Hsp60 malfunction on the quantitative and qualitative composition of the mitochondrial proteome.

References:

1. Magen, D. et al. (2008) Am. J. Hum. Genet. 83, 30-42

2. Hansen, J. J. et al. (2002) Am. J. Hum. Genet. 70, 1328-1332

3. Hansen, J. et al. (2007) J. Neurol. 254, 897-900

4. Bross, P. et al. (2008) J. Biol. Chem. 283, 15694-15700

5. Christensen, J. H. et al. (2010) Cell Stress. Chaperones.