Intermittent enzyme replacement therapy with recombinant human ß-galactosidase prevents neuraminidase 1 deficiency

Mutations in the galactosidase ß 1 (GLB1) gene cause lysosomal ß-galactosidase (ß-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. ß-Gal and neuraminidase 1 (NEU1) form a multienzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, ß-Gal only partially depends on PPCA, prompting us to investigate the role that ß-Gal plays in the multienzyme complex. Here, we demonstrate that ß-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human ß-Gal (rhß-Gal) or chronic lentiviral-mediated GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy dosing with rhß-Gal, followed by enzyme withdrawal, is sufficient to augment ß-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of ß-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular dosing with rhß-Gal. Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of ß-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent intracerebroventricular enzyme replacement therapy dosing with rhß-Gal is a tunable approach that can safely augment ß-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain.

Intracerebroventricular enzyme replacement therapy with ß-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Autosomal recessive mutations in the galactosidase ß1 (GLB1) gene cause lysosomal ß-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human ß-gal (rhß-gal) produced in Chinese hamster ovary cells enabled direct and precise rhß-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhß-gal was sufficient for normalizing ß-gal activity and mediating substrate clearance for several weeks. We found that rhß-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor-mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhß-gal (100 µg) resulted in broad bilateral biodistribution of rhß-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhß-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of ß-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhß-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) phosphorylation by protein kinase Cδ (PKCδ) inhibits mitochondria elimination by lysosomal-like structures following ischemia and reoxygenation-induced injury.

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis.

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.

Abl kinases are required for invadopodia formation and chemokine-induced invasion.

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.

Identifying therapeutic drug targets using bidirectional effect genes.

Prioritizing genes for translation to therapeutics for common diseases has been challenging. Here, we propose an approach to identify drug targets with high probability of success by focusing on genes with both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). We find 98 BEST genes for a variety of indications. Drugs targeting those genes are 3.8-fold more likely to be approved than non-BEST genes. We focus on five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants in those genes result in significantly increased risk for idiopathic short stature (ISS) (OR = 2.75, p = 3.99 × 10-8). Finally, using functional experiments, we demonstrate that adding an exogenous CNP analog (encoded by NPPC) rescues the phenotype, thus validating its potential as a therapeutic treatment for ISS. Our results show the value of looking for bidirectional effects to identify and validate drug targets.

Regulation of mitochondrial processes: a target for heart failure.

Cardiac mitochondria, the main source of energy as well as free radicals, are vital organelles for normal functioning of the heart. Mitochondrial number, structure, turnover and function are regulated by processes such as mitochondrial protein quality control, mitochondrial fusion and fission and mitophagy. Recent studies suggest that abnormal changes in these mitochondrial regulatory processes may contribute to the pathology of heart failure (HF). Here we discuss these processes and their potential as therapeutic targets.

Differential Uptake of NAGLU-IGF2 and Unmodified NAGLU in Cellular Models of Sanfilippo Syndrome Type B.

Sanfilippo syndrome type B, or mucopolysaccharidosis IIIB (MPS IIIB), is a rare autosomal recessive lysosomal storage disease caused by a deficiency of α-N-acetylglucosaminidase (NAGLU). Deficiency in NAGLU disrupts the lysosomal turnover of heparan sulfate (HS), which results in the abnormal accumulation of partially degraded HS in cells and tissues. BMN 250 (NAGLU-insulin-like growth factor 2 [IGF2]) is a recombinant fusion protein developed as an investigational enzyme replacement therapy for MPS IIIB. The IGF2 peptide on BMN 250 promotes enhanced targeting of the enzyme to lysosomes through its interaction with the mannose 6-phosphate receptor. The focus of these studies was to further characterize the ability of NAGLU-IGF2 to clear accumulated HS compared to unmodified NAGLU in primary cellular models of MPS IIIB. Here, we establish distinct primary cell models of MPS IIIB with HS accumulation. These cellular models revealed distinct NAGLU uptake characteristics that depend on the duration of exposure. We found that with sustained exposure, NAGLU uptake and HS clearance occurred independent of known lysosomal targeting signals. In contrast, under conditions of limited exposure duration, NAGLU-IGF2 was taken up more rapidly than the unmodified NAGLU into MPS IIIB primary fibroblasts, astrocytes, and cortical neurons, where it efficiently degraded accumulated HS. These studies illustrate the importance of using physiologically relevant conditions in the evaluation of enzyme replacement therapies in cellular models.