Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Cross-diffusion induced patterns for a single-step enzymatic reaction

Several different enzymes display an apparent diffusion coefficient that increases with the concentration of their substrate. Moreover, their motion becomes directed in substrate gradients. Currently, there are several competing models for these transport dynamics. Here, we use mathematical modeling and numerical simulations to analyze whether the enzymatic reactions can generate a significant feedback from enzyme transport onto the substrate profile. We find that this feedback can generate spontaneous spatial patterns in the enzyme distribution, with just a single-step catalytic reaction. However, patterns are formed only for a subclass of transport models. For such models, nonspecific repulsive interactions between the enzyme and the substrate, or attractive interactions between the enzyme and the product, cause the enzyme to accumulate in regions of low substrate concentration. Reactions then amplify local substrate and product fluctuations, causing enzymes to further accumulate where substrate is low. Experimental analysis of this pattern formation process could discriminate between different transport models.

Alterations in EGF-Endocytosis, Lysosomal Enzyme Transport and Maturation of Cathepsins in Juvenile Neuronal Ceroid Lipofuscinosis Fibroblasts

Background: Juvenile neuronal ceroid lipofuscinosis (JNCL), one of the most frequent forms of the NCL storage diseases, is known to be caused by loss-of-function mutations in ceroid-lipofuscinosis neuronal protein 3 (CLN3), but its cell function has not been fully elucidated. We previously reported increased lysosomal pH in CLN3 deficient cells. In the present study, we analysed the consequences of this effect in the endo-lysosomal pathways in CLN3 cells. Methods: The present study investigated different endo-lysosomal pathways in control, CLN2, CLN3 human skin fibroblasts under high and low proteolysis conditions. Cell surface biotinylation after EGF (2 ng/mL) stimulation, EGF phosphorylation (Tyr-845), retromer and cation-independent mannose-6- phosphate receptor (CI-MPR) levels and stability, EGF degradation pathways and cathepsin L and D levels were analysed by western blots. Caveolae mediated endocytosis was analysed by flow cytometry. CIMPR subcellular localization was ascertained by immunocytochemistry, confocal microscopy and further image analysis. Results: Whereas caveolae-mediated endocytosis was not affected in CLN3 cells, clathrin-mediated epidermal growth factor (EGF) internalization was reduced, along with EGF receptor (EGFR) phosphorylation. In addition, cell surface EGFR levels and recycling to the cell membrane were increased. EGFR lysosomal degradation was impaired and our results suggest that the receptor was diverted to proteasomal degradation. We also analysed the machinery responsible for lysosomal hydrolase transport to the lysosome and found increased stability of CIMPR, a major receptor implicated in the transport of hydrolases. The subcellular distribution of the CI-MPR was also altered in CLN3 cells, since it accumulated within the Trans-Golgi network (TGN) and did not progress into the lysosomes. In addition, we found a reduced turnover of retromer subunits, a complex that retrieves the CI-MPR from endosomes to the TGN. Finally and as a possible consequence of these alterations in lysosomal enzyme transport, cathepsin L and D maturation were found suppressed in CLN3 cells. Conclusion: Altogether, these results point to increased lisosomal pH as a pivotal event causing various alterations in intracellular traffic associated to the development of JNCL disease.

Pharmacologic manipulation of lysosomal enzyme transport across the blood–brain barrier

The adult blood–brain barrier, unlike the neonatal blood–brain barrier, does not transport lysosomal enzymes into brain, making enzyme replacement therapy ineffective in treating the central nervous system symptoms of lysosomal storage diseases. However, enzyme transport can be re-induced with alpha-adrenergics. Here, we examined agents that are known to alter the blood–brain barrier transport of large molecules or to induce lysosomal enzyme transport across the blood–brain barrier ((±)epinephrine, insulin, retinoic acid, and lipopolysaccharide) in 2-week-old and adult mice. In 2-week-old adolescent mice, all these pharmacologic agents increased brain and heart uptake of phosphorylated human β-glucuronidase. In 8-week-old adult mice, manipulations with (±)epinephrine, insulin, and retinoic acid were significantly effective on uptake by brain and heart. The increased uptake of phosphorylated human β-glucuronidase was inhibited by mannose 6-phosphate for the agents (±)epinephrine and retinoic acid and by L-NG-nitroarginine methyl ester for the agent lipopolysaccharide in neonatal and adult mice. An in situ brain perfusion study revealed that retinoic acid directly modulated the transport of phosphorylated human β-glucuronidase across the blood–brain barrier. The present study indicates that there are multiple opportunities to at least transiently induce phosphorylated human β-glucuronidase transport at the adult blood–brain barrier.

Genetic Diseases That Predispose to Early Liver Cirrhosis

Inherited liver diseases are a group of metabolic and genetic defects that typically cause early chronic liver involvement. Most are due to a defect of an enzyme/transport protein that alters a metabolic pathway and exerts a pathogenic role mainly in the liver. The prevalence is variable, but most are rare pathologies. We review the pathophysiology of such diseases and the diagnostic contribution of laboratory tests, focusing on the role of molecular genetics. In fact, thanks to recent advances in genetics, molecular analysis permits early and specific diagnosis for most disorders and helps to reduce the invasive approach of liver biopsy.