Clathrin-Mediated Endocytosis Is Impaired in Type A–B Niemann–Pick Disease Model Cells and Can Be Restored by ICAM-1-Mediated Enzyme Replacement

Abstract

Drugs often use endocytosis to achieve intracellular delivery, either by passive uptake from the extracellular fluid or by active targeting of cell surface features such as endocytic receptors. An example is enzyme replacement therapy, a clinically practiced treatment for several lysosomal storage diseases where glycosylated recombinant enzymes naturally target the mannose-6-phosphate receptor and are internalized by clathrin mediated endocytosis (CME). However, lysosomal substrate accumulation, a hallmark of these diseases, has been indirectly linked to aberrant endocytic activity. These effects are poorly understood, creating an obstacle to therapeutic efficiency. Here we explored endocytic activity in fibroblasts from patients with type A Niemann–Pick disease, a lysosomal storage disease characterized by acid sphingomyelinase (ASM) deficiency. The uptake of fluid phase markers and clathrin-associated ligands, formation of endocytic structures, and recruitment of intracellular clathrin to ligand binding sites were all altered, demonstrating aberrant CME in these cells. Model polymer nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1), which are internalized by a clathrin-independent route, enhanced the intracellular delivery of recombinant ASM more than 10-fold compared to free enzyme. This strategy reduced substrate accumulation and restored clathrin endocytic activity to wild-type levels. There appears to be a relationship between lysosomal storage and diminished CME, and bypassing this pathway by targeting ICAM-1 may enhance future therapies for lysosomal storage diseases.

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

“…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.…”

“…We defined cell-surface caveoli as invaginations on the plasmalemma with a size ≤ 50 nm in diameter and void of an electron-light halo surrounding the opening of the invagination (this halo is characteristic of clathrin-coated pits) 30 . Semiquantitative analysis was performed to analyze the number of invaginations per cell-surface area.…”

“…However, several groups have observed alterations in the behavior of endocytic receptors and pathways in LSDs 22-27 . These alterations impact intracellular delivery and effects of therapeutic enzymes 28-31 , such as the case of acid sphingomyelinase (ASM) required for treatment of type A Niemann-Pick disease (NPD) 28-30 .…”

ICAM-1 targeting, intracellular trafficking, and functional activity of polymer nanocarriers coated with a fibrinogen-derived peptide for lysosomal enzyme replacement

“…For the reasons already described in the introduction, NC surface-loading of lysosomal enzymes is reasonable. However, ASM (as other lysosomal enzymes) displays M6P residues [2,35] which may compete for binding to the M6P receptor expressed on most cells, and uptake via the M6P receptor has been shown to be altered in NPD [36,37]. Under this scenario, that fact that γ3 NCs bound well to cells did not guarantee that γ3/ASM NCs would, as the presence of ASM on γ3 NCs may have obliterated the delivery improvement originally intended.…”