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 autophagy 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 in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. 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.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.

AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease.

Neuronal ceroid lipofuscinosis type 7 (CLN7) disease is a lysosomal storage disease caused by mutations in the facilitator superfamily domain containing 8 (MFSD8) gene, which encodes a membrane-bound lysosomal protein, MFSD8. To test the effectiveness and safety of adeno-associated viral (AAV) gene therapy, an in vitro study demonstrated that AAV2/MFSD8 dose dependently rescued lysosomal function in fibroblasts from a CLN7 patient. An in vivo efficacy study using intrathecal administration of AAV9/MFSD8 to Mfsd8- /- mice at P7-P10 or P120 with high or low dose led to clear age- and dose-dependent effects. A high dose of AAV9/MFSD8 at P7-P10 resulted in widespread MFSD8 mRNA expression, tendency of amelioration of subunit c of mitochondrial ATP synthase accumulation and glial fibrillary acidic protein immunoreactivity, normalization of impaired behaviors, doubled median life span, and extended normal body weight gain. In vivo safety studies in rodents concluded that intrathecal administration of AAV9/MFSD8 was safe and well tolerated. In summary, these results demonstrated that the AAV9/MFSD8 vector is both effective and safe in preclinical models.

Rescue of α-synuclein aggregation in Parkinson's patient neurons by synergistic enhancement of ER proteostasis and protein trafficking.

Neurodegenerative disorders are characterized by a collapse in proteostasis, as shown by the accumulation of insoluble protein aggregates in the brain. Proteostasis involves a balance of protein synthesis, folding, trafficking, and degradation, but how aggregates perturb these pathways is unknown. Using Parkinson's disease (PD) patient midbrain cultures, we find that aggregated α-synuclein induces endoplasmic reticulum (ER) fragmentation and compromises ER protein folding capacity, leading to misfolding and aggregation of immature lysosomal ß-glucocerebrosidase. Despite this, PD neurons fail to initiate the unfolded protein response, indicating perturbations in sensing or transducing protein misfolding signals in the ER. Small molecule enhancement of ER proteostasis machinery promotes ß-glucocerebrosidase solubility, while simultaneous enhancement of trafficking improves ER morphology, lysosomal function, and reduces α-synuclein. Our studies suggest that aggregated α-synuclein perturbs the ability of neurons to respond to misfolded proteins in the ER, and that synergistic enhancement of multiple proteostasis branches may provide therapeutic benefit in PD.

Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by α-Synuclein.

Age-related neurodegenerative disorders are characterized by a slow, persistent accumulation of aggregated proteins. Although cells can elicit physiological responses to enhance cellular clearance and counteract accumulation, it is unclear how pathogenic proteins evade this process in disease. We find that Parkinson's disease α-synuclein perturbs the physiological response to lysosomal stress by impeding the SNARE protein ykt6. Cytosolic ykt6 is normally autoinhibited by a unique farnesyl-mediated regulatory mechanism; however, during lysosomal stress, it activates and redistributes into membranes to preferentially promote hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, thereby disabling the lysosomal stress response and facilitating protein accumulation. Activating ykt6 by small-molecule farnesyltransferase inhibitors restores lysosomal activity and reduces α-synuclein in patient-derived neurons and mice. Our findings indicate that α-synuclein creates a permissive environment for aggregate persistence by inhibiting regulated cellular clearance and provide a therapeutic strategy to restore protein homeostasis by harnessing SNARE activity.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Reversible Conformational Conversion of α-Synuclein into Toxic Assemblies by Glucosylceramide.

α-Synuclein (α-syn) aggregation is a key event in Parkinson's disease (PD). Mutations in glycosphingolipid (GSL)-degrading glucocerebrosidase are risk factors for PD, indicating that disrupted GSL clearance plays a key role in α-syn aggregation. However, the mechanisms of GSL-induced aggregation are not completely understood. We document the presence of physiological α-syn conformers in human midbrain dopamine neurons and tested their contribution to the aggregation process. Pathological α-syn assembly mainly occurred through the conversion of high molecular weight (HMW) physiological α-syn conformers into compact, assembly-state intermediates by glucosylceramide (GluCer), without apparent disassembly into free monomers. This process was reversible in vitro through GluCer depletion. Reducing GSLs in PD patient neurons with and without GBA1 mutations diminished pathology and restored physiological α-syn conformers that associated with synapses. Our work indicates that GSLs control the toxic conversion of physiological α-syn conformers in a reversible manner that is amenable to therapeutic intervention by GSL reducing agents.

Pregnancy-induced amelioration of muscular dystrophy phenotype in mdx mice via muscle membrane stabilization effect of glucocorticoid.

Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, mdx mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from mdx mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into mdx mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in mdx mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies.