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

<i>Annexin A1</i> (<i>Anxa1</i>) is up-regulated after treatment with corticosterone.

<p>(A, B) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle, corticosterone or corticosterone and mifepristone. After 1 hour of incubation with 100% medium or 50% hypo-osmotic shock medium, relative dead cells were calculated as Trypan blue-positive cells (red arrows) per total cells (n = 3). Bar (200 μm). (C) mRNA levels of <i>Inta7</i>, <i>MG53</i>, <i>Anxa1</i>, and <i>Serca1</i> in vehicle or corticosterone-treated wild-type or <i>mdx</i> myotubes were quantified with real-time qPCR (n = 3). <i>β-Actin</i> was used for internal control. (D) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle or corticosterone, and stained for MHC (green) and annexin A1 (red). DAPI (blue) denotes all nuclei. Bar (50 μm).</p

Pregnancy-Induced Amelioration of Muscular Dystrophy Phenotype in <i>mdx</i> Mice via Muscle Membrane Stabilization Effect of Glucocorticoid

<div><p>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, <i>mdx</i> 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 <i>mdx</i> 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 <i>mdx</i> mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in <i>mdx</i> 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.</p></div

Muscle fiber characterizations in the diaphragm from virgin, pregnant and postpartum <i>mdx</i> mice.

<p>(A) Diaphragm from 2.5-month-old virgin and pregnant female <i>mdx</i> mice with EBD injection. Gestation day 7.5 (GD7.5) and GD14.5 mean 7 and 14 days after the recognition of vaginal plug in mated female mice, respectively. Postpartum day 2 (PD2), PD7 and PD14 mean 2, 7 and 14 days after parturition, respectively. Bar (2 mm) (B) Diaphragm sections examined by fluorescence microscopy. Bars (200 μm). (C) The EBD uptake at GD14.5 (n = 3), PD2 (n = 4), PD7 (n = 3) and the virgin female <i>mdx</i> mice (n = 5). (D) Fiber size (μm) distribution in virgin (n = 4) and PD2 <i>mdx</i> mice (n = 4). (E) Histogram of number of fibers with centrally located nuclei (CLN) in virgin (n = 9), PD2 (n = 7) and PD7 (n = 5) <i>mdx</i> mice.</p

Decreased EBD uptake and increased annexin A1 up-regulation in the diaphragm of male <i>mdx</i> mice after injection with corticosterone or PD2 female <i>mdx</i> mice.

<p>EBD-positive area per total diaphragm section area was calculated after injecting male (A and B) and female (C) <i>mdx</i> mice with vehicle, corticosterone, estradiol, or progesterone for 2 days. Vehicle, n = 8 (male) and n = 4 (female); corticosterone, n = 9 (male) and n = 5 (female); estradiol, n = 9 (male) and n = 5 (female); progesterone, n = 9 (male) and n = 5 (female). Bar (200 μm). (D) mRNA levels of <i>Anxa1</i> in vehicle or corticosterone-treated mice, or virgin or PD2 female mice were quantified with real-time qPCR. Vehicle, n = 4 (male) and n = 6 (female); corticosterone, n = 6 (male) and n = 6 (female); n = 7 (virgin) and n = 6 (PD2). <i>GAPDH</i> was used for internal control.</p

Over-expression of annexin A1 can protect muscle fiber death in hypo-osmotic shock.

<p>(A) Female <i>mdx</i> myoblasts were infected with empty retroviral vector (pMX), annexin A1 retroviral vector (pMX-Anxa1), control lentiviral shRNA vector or annexin A1 lentiviral shRNA vector. After myotube formation, cells were stained for MHC (green) and annexin A1 (red). DAPI (blue) denotes all nuclei. Bar (50 μm). Relative mRNA level of <i>annexin A1</i> in <i>mdx</i> myotubes was also quantified with real-time qPCR after viral infection (n = 3). <i>β-Actin</i> was used for internal control. (B) Myotubes were incubated with 100% medium or 50% hypo-osmotic shock medium for 1 hour, and stained with Trypan blue (red arrows). Relative dead cells were calculated as Trypan blue-positive cells per total cells (n = 3). Bar (200 μm).</p

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&nbsp;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.)