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Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates.

ABSTRACT: Lysosomal dysfunction has been implicated both pathologically and genetically in neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease (PD). Lysosomal gene deficiencies cause lysosomal storage disorders, many of which involve neurodegeneration. Heterozygous mutations of some of these genes, such as GBA1, are associated with PD. CTSD is the gene encoding Cathepsin D (CTSD), a lysosomal protein hydrolase, and homozygous CTSD deficiency results in neuronal ceroid-lipofuscinosis, which is characterized by the early onset, progressive neurodegeneration. CTSD deficiency was also associated with deposition of α-synuclein aggregates, the hallmark of PD. However, whether partial deficiency of CTSD has a role in the late onset progressive neurodegenerative disorders, including PD, remains unknown. Here, we generated cell lines harboring heterozygous nonsense mutations in CTSD with genomic editing using the zinc finger nucleases. Heterozygous mutation in CTSD resulted in partial loss of CTSD activity, leading to reduced lysosomal activity. The CTSD mutation also resulted in increased accumulation of intracellular α-synuclein aggregates and the secretion of the aggregates. When α-synuclein was introduced in the media, internalized α-synuclein aggregates accumulated at higher levels in CTSD+/- cells than in the wild-type cells. Consistent with these results, transcellular transmission of α-synuclein aggregates was increased in CTSD+/- cells. The increased transmission of α-synuclein aggregates sustained during the successive passages of CTSD+/- cells. These results suggest that partial loss of CTSD activity is sufficient to cause a reduction in lysosomal function, which in turn leads to α-synuclein aggregation and propagation of the aggregates.

SUBMITTER: Bae EJ

PROVIDER: S-EPMC4632307 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Project description:Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Project description:Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer's proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases.

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Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates.

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