Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies observed in the spinal motor neurons of sporadic ALS and is decreased in the cerebrospinal fluid of ALS patients. Despite prominent deposition of CysC in ALS, the roles of CysC in the central nervous system remain unknown. Here, we identified the neuroprotective activity of CysC against ALS-linked mutant Cu/Zn-superoxide dismutase (SOD1)-mediated toxicity. We found that exogenously added CysC protected neuronal cells including primary cultured motor neurons. Moreover, the neuroprotective property of CysC was dependent on the coordinated activation of two distinct pathways: autophagy induction through AMPK-mTOR pathway and inhibition of cathepsin B. Furthermore, exogenously added CysC was transduced into the cells and aggregated in the cytosol under oxidative stress conditions, implying a relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest CysC is an endogenous neuroprotective agent and targeting CysC in motor neurons may provide a novel therapeutic strategy for ALS.Failure of protein quality control and degradation is deeply involved in the pathomechanisms of neurodegenerative diseases. Prominent deposition of disease-specific proteins is characteristic in neurodegenerative diseases, such as amyloid-β in Alzheimer''s disease or huntingtin in Huntington''s disease. Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by the selective loss of motor neurons. While 90% of ALS is sporadic, 10% is inherited. Among the inherited ALS cases, dominant mutations in Cu/Zn superoxide dismutase (SOD1) are the frequent cause of inherited ALS.¹ Transgenic mice and rats expressing a human gene for SOD1 with an ALS-linked mutation develop an ALS phenotype, whereas those with deletion of wild-type SOD1 do not, indicating that acquired toxicity mediated by mutant SOD1 is involved in neurodegeneration.^2,3 In SOD1-linked ALS, SOD1-containing inclusions or oligomerized protein complexes have been specifically found in the spinal motor neurons and astrocytes.⁴ It has been proposed that mutant SOD1 proteins are misfolded and consequently aggregated, gaining toxic properties at some stage in their formation.⁵ Furthermore, recent studies have suggested that the accumulation of misfolded SOD1 proteins is involved in the pathomechanisms of sporadic ALS.^6,7 Therefore, a reduction of misfolded SOD1 proteins might be one of the viable therapeutic approaches for ALS.Cystatin C (CysC) is an endogenous cysteine protease inhibitor and expressed in various tissues.⁸ In the central nervous system, CysC is mainly secreted from the choroid plexus into the cerebrospinal fluid. CysC is a member of the type-II Cystatin family and inhibits cathepsin B, S and F.⁹ Although its precise function, especially in the central nervous system, is still uncertain, some studies have revealed that CysC has a neuroprotective role in neurodegenerative diseases.¹⁰ In a mouse model for Alzheimer''s disease, overexpression of human CysC in the mice reduced deposits of amyloid-β fibrils.¹¹ CysC has been shown to improve the survival of dopaminergic neurons in a rat model of Parkinson''s disease.¹² In sporadic ALS, CysC is a major component of Bunina bodies, which are ALS-specific inclusion bodies, found in remaining motor neurons,¹³ and the levels of CysC are decreased in the cerebrospinal fluid of ALS patients.^14,15 Intriguingly, it was also reported that the concentration of CysC in the cerebrospinal fluid is correlated with the survival time of ALS patients,¹⁵ implying a potent neuroprotective property of CysC in ALS.Previous reports showed that CysC induces autophagy to protect neuronal cells against various stresses including serum or growth-factor deprivation and oxidative stresses.^10,16 Autophagy is a major intracellular proteolytic pathway that targets misfolded or aggregated proteins as well as the ubiquitin-proteasome pathway. Because the ubiquitin-proteasome pathway is impaired in both SOD1-linked^17,18 and SOD1-unrelated^19,20 ALS models, autophagy activation may complementally degrade the abnormal proteins to rescue motor neurons. Indeed, involvement of autophagy is implicated in the experimental models of ALS.^21,22 Moreover, recent studies have shown that cathepsin B (CatB), a member of the cysteine protease family that is inhibited by CysC, is deeply involved in motor neuronal degeneration. Increased immunoreactivity of CatB was often found in the neurons of sporadic ALS patients²³ or ALS model mice²⁴ and CatB-knockout mice showed a lower rate of motor neuron death after nerve injury,²⁵ suggesting that inhibition of CatB is beneficial for motor neuronal survival. These previous data suggest the possibility that CysC is a promising therapeutic candidate for ALS. However, no evidence has been provided for the role of CysC in neuroprotection in ALS models.Here, we performed direct tests of the neuroprotective property of CysC using neuroblastoma cell Neuro2a (N2a) and primary mix-cultured motor neurons derived from mutant SOD1 transgenic mice and identified that CysC is a novel neuroprotective agent against mutant SOD1-mediated neurotoxicity that acts through induction of autophagy and inhibition of CatB.