Corrigendum: Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties

Cathepsin D (CTSD) is a lysosomal protease important for the degradation of various substrates, including disease-associated proteins like α-synuclein (a-syn), amyloid precursor protein (APP) and tau, all of which tend to aggregate if not efficiently degraded. Hence, it is not surprising that genetic variants within the CTSD gene have been linked to neurodegenerative diseases, like Parkinson's and Alzheimer's disease (PD, AD), as well as the lysosomal storage disorder neuronal ceroid lipofuscinosis type-10 (NCL10). Although recent studies have shown the molecular dependence of substrate degradation via CTSD within autophagic pathways, only little is known about the precise role of lysosomal CTSD function in disease development. We here performed biochemical, cellular and structural analyses of eleven disease-causing CTSD point mutations found in genomic sequencing data of patients to understand their role in neurodegeneration. These CTSD variants were analyzed for cellular localization, maturation and enzymatic activity in overexpression analyses. Moreover, for PD-associated mutants, intracellular degradation of a-syn was monitored. In summary, our results suggest that NCL10-associated CTSD variants are significantly impaired in lysosomal maturation and enzymatic activity, whereas the AD- and PD-associated variants seemed rather unaffected, indicating normal maturation, and lysosomal presence. Interestingly, a PD-associated CTSD variant (A239V) exhibited increased enzymatic activity accompanied by enhanced a-syn degradation. By structural analyses of this mutant utilizing molecular dynamics simulation (MDS), we identified a structural change within a loop adjacent to the catalytic center leading to a higher flexibility and potentially accelerated substrate exchange rates. Our data sheds light onto the role of CTSD in disease development and helps to understand the structural regulation of enzymatic function, which could be utilized for targeted CTSD activation. Because of the degradative function of CTSD, this enzyme is especially interesting for therapeutic strategies tackling protein aggregates in neurodegenerative disorders

Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties

Cathepsin D (CTSD) is a lysosomal protease important for the degradation of various substrates, including disease-associated proteins like α-synuclein (a-syn), amyloid precursor protein (APP) and tau, all of which tend to aggregate if not efficiently degraded. Hence, it is not surprising that genetic variants within the CTSD gene have been linked to neurodegenerative diseases, like Parkinson’s and Alzheimer’s disease (PD, AD), as well as the lysosomal storage disorder neuronal ceroid lipofuscinosis type-10 (NCL10). Although recent studies have shown the molecular dependence of substrate degradation via CTSD within autophagic pathways, only little is known about the precise role of lysosomal CTSD function in disease development. We here performed biochemical, cellular and structural analyses of eleven disease-causing CTSD point mutations found in genomic sequencing data of patients to understand their role in neurodegeneration. These CTSD variants were analyzed for cellular localization, maturation and enzymatic activity in overexpression analyses. Moreover, for PD-associated mutants, intracellular degradation of a-syn was monitored. In summary, our results suggest that NCL10-associated CTSD variants are significantly impaired in lysosomal maturation and enzymatic activity, whereas the AD- and PD-associated variants seemed rather unaffected, indicating normal maturation, and lysosomal presence. Interestingly, a PD-associated CTSD variant (A239V) exhibited increased enzymatic activity accompanied by enhanced a-syn degradation. By structural analyses of this mutant utilizing molecular dynamics simulation (MDS), we identified a structural change within a loop adjacent to the catalytic center leading to a higher flexibility and potentially accelerated substrate exchange rates. Our data sheds light onto the role of CTSD in disease development and helps to understand the structural regulation of enzymatic function, which could be utilized for targeted CTSD activation. Because of the degradative function of CTSD, this enzyme is especially interesting for therapeutic strategies tackling protein aggregates in neurodegenerative disorders

Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagic as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a CTSD-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance, but also restored endo-lysosome and autophagy function in human and murine neurons as well as tissue. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.authorsversionepub_ahead_of_prin

Toxic Metamorphosis—How Changes from Lysosomal to Cytosolic pH Modify the Alpha-Synuclein Aggregation Pattern

Alpha-synuclein (aSyn) is a cytosolic, aggregation-prone protein that is associated with neurodegenerative disorders like Parkinson’s disease. Interestingly, the protein can appear in different conformations, including monomeric and oligomeric forms as well as amyloid fibrils. Its individual structural constituents seem to be dependent on various factors and the composition of the respective cellular surroundings. Although under physiological conditions, most aSyn is found in the cytosol and synapses of neurons, aSyn can also be found in lysosomal compartments, where it gets degraded. We here compare the assembly speed, morphology, folding state, and spreading of aSyn at cytosolic pH (pH 7.4) and lysosomal pH (pH 5) using Thioflavin T, transmission electron microscopy, circular dichroism, and Fourier transform infrared spectroscopy. Interestingly, we found substantial differences between aSyn aggregation under neutral and acidic pH conditions, like those present in cytosolic and lysosomal cellular compartments. Also, lysosomal aSyn enriched from an aSyn-overexpressing cell line was able to seed aggregation in a concentration-dependent manner. Moreover, we observed that aSyn aggregates formed under in vitro lysosomal pH (pH 5) conditions were not stable at neutral pH and collapsed into partly soluble aggregates with changed structural characteristics. Our findings have meaningful implications in intracellular toxicity events as well as in lysis procedures for molecular and structural characterization of intracellular aSyn conformers.<br/