Altered Clathrin-Independent Endocytosis in Type A Niemann-Pick Disease Cells and Rescue by ICAM-1-Targeted Enzyme Delivery

Abstract

Pharmaceutical intervention often requires therapeutics and/or their carriers to enter cells via endocytosis. Therefore, endocytic aberrancies resulting from disease represent a key, yet often overlooked, parameter in designing therapeutic strategies. In the case of lysosomal storage diseases (LSDs), characterized by lysosomal accumulation of undegraded substances, common clinical interventions rely on endocytosis of recombinant enzymes. However, the lysosomal defect in these diseases can affect endocytosis, as we recently demonstrated for clathrin-mediated uptake in patient fibroblasts with type A Niemann-Pick disease (NPD), a disorder characterized by acid sphingomylinase (ASM) deficiency and subsequent sphingomyelin storage. Using similar cells, we have examined if this is also the case for clathrin-independent pathways, including caveolae-mediated endocytosis and macropinocytosis. We observed impaired caveolin-1 enrichment at ligand-binding sites in NPD vs. wild type fibroblasts, corresponding with altered uptake of ligands and fluid-phase markers by both pathways. Similarly, aberrant lysosomal storage of sphingomyelin induced by pharmacological means also diminished uptake. Partial degradation of the lysosomal storage by untargeted recombinant ASM lead to partial uptake enhancement, while both parameters were restored to wild type levels by ASM delivery using model polymer nanocarriers specifically targeted to intercellular adhesion molecule-1 (anti-ICAM NCs). Carriers also restored caveolin-1 enrichment at ligand-binding sites and uptake through the caveolar and macropinocytic routes. These results demonstrate a link between lysosomal storage in NPD and alterations in clathrin-independent endocytosis, which could apply to other LSDs. Hence, this study shall guide the design of therapeutic approaches using viable endocytic pathways.

A Comparative Study on the Alterations of Endocytic Pathways in Multiple Lysosomal Storage Disorders

“…In this particular case, the validation described above indicates that these two routes contributed similarly to dextran uptake in wild-type fibroblasts. This parallels our previous data 29, 30 and was also confirmed here (left bars in Fig. 2b and 2c), showing that dextran uptake was diminished by 43% and 44% in the presence of MDC or filipin, which respectively inhibit the clathrin and caveolar routes.…”

“…Over the course of one hour, all fibroblast types accumulated fewer dextran-filled compartments than wild-type cells, as observed by fluorescence microscopy (Fig. 1a), consistent with our previous findings in NPD cells 29, 30 . Automatic image analysis revealed ~40% fewer dextran-positive vesicles in diseased cells vs. wild-type cells (Fig.…”

“…We recently demonstrated that lysosomal storage in NPD impairs bulk pinocytosis 29, 30 . To examine if this is also the case for other LSDs, we compared the uptake of Texas Red dextran, a fluid-phase marker, in fibroblasts from patients of NPC, Gaucher, and Fabry diseases (Fig.…”

“…As described above, samples were analyzed by fluorescence microscopy, whereby surface Tf or CTB appear yellow vs. internalized counterparts that appear green 29, 30 . The percentage of internalized green fluorescent pixels over the total cell-associated fluorescence, and the number of green fluorescent vesicles per cell were quantified, where the cell borders were obtained by phase-contrast microscopy 29, 30 . LSD cells were compared to wild-type cells, and control conditions were compared to inhibitor conditions, as described.…”

“…These are prototype carriers, with coating efficacy, in vivo targeting, and intracellular transport comparable to anti-ICAM NCs made of biodegradable poly(lactic-co-glycolic acid) 42, 43 , therefore providing a valid model for cell culture assays. In addition, the specificity of anti-ICAM NCs (vs. control IgG NCs) in terms of targeting, endocytic uptake, and lysosomal delivery has been extensively demonstrated in both cell cultures and mouse models 29, 37, 39-43 .…”