Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons

Abstract

Aspartylglucosaminuria (AGU) is a neurodegenerative lysosomal storage disease that is caused by mutations in the gene encoding for a soluble hydrolase, aspartylglucosaminidase (AGA). In this study, we have used our recently developed mouse model for AGU and analyzed processing, intracellular localization, and endocytosis of recombinant AGA in telencephalic AGU mouse neurons in vitro. The processing steps of AGA were found to be similar to the peripheral cells, but both the accumulation of the inactive precursor molecule and delayed lysosomal processing of the enzyme were detected. AGA was distributed to the cell soma and neuronal processes but was not found in the nerve terminals. Endocytotic capability of cultured telencephalic neurons was comparable to that of fibroblasts, and endocytosis of AGA was blocked by free mannose-6-phosphate (M6P), indicating that uptake of the enzyme was mediated by M6P receptors (M6PRs). Uptake of extracellular AGA was also studied in the tumor-derived cell lines rat pheochromocytoma (PC12) and mouse neuroblastoma cells (N18), which both endocytosed AGA poorly as compared with cultured primary neurons. Expression of cationindependent M6PRs (CI-M6PRs) in different cell lines correlated well with the endocytotic capability of these cells. Although a punctate expression pattern of CI-M6PRs was found in fibroblasts and cultured primary neurons, the expression was beyond the detection limit in PC12 and N18 cells. This indicates that PC12 and N18 are not feasible cell lines to describe neuronal uptake of mannose-6-phosphate-tagged proteins. This in vitro data will form an important basis for the braintargeted therapy of AGU.

Degradation of fibrillar forms of Alzheimer's amyloid β-peptide by macrophages

“…As shown in Figure 2, incubation with mannose-6-phosphorylated lysosomal enzymes increased the ability of microglia to degrade fAβ, as compared with parallel untreated control cells. Because the mannose phosphorylated at the 6 position is the entity recognized by the receptor, uptake of lysosomal enzymes can be inhibited by excess mannose-6 phosphate [14,15]. When the enzyme incubation was done in the presence of 10mM mannose-6 phosphate to compete for receptor binding, microglia failed to degrade fAβ.…”

Aspartylglucosaminidase (AGA) is efficiently produced and endocytosed by glial cells: implication for the therapy of a lysosomal storage disorder

“…The glycosylation and phosphorylation of AGA are well known processes and the three-dimensional structure of the enzyme with the specific activation sites has been characterized in detail [6,28]. The processing of AGA into the subunits has been studied in several cell lines revealing an exceptionally prolonged processing in neurons and in brain tissue [11,23]. Here we demonstrated a similar delayed processing in neurons using the NSE and AGA promoters to mediate expression of AGA.…”

“…Previous data have indicated a delay in the maturation of AGA in neuronal cell cultures [11] and also in the total brain tissue of mice [23]. Proteolytic processing of AGA is essential for enzymatic activity.…”

“…The endocytosis of AGA in the neurons has been previously studied [11]. The presence of mannose-6-phosphate receptors in neurons and glia [12] provides the possibility to correct brain pathology in lysosomal storage disorders.…”

Use of nonviral promoters in adenovirus‐mediated gene therapy: reduction of lysosomal storage in the aspartylglucosaminuria mouse

“…This is a prerequisite for successful therapeutic approaches for treating lysosomal storage disorders affecting the CNS [15]. Indeed, adenoviral injection into the lateral ventricles of AGU mice led to the migration of AGA enzyme also to the brain tissue and partial elimination of the storage vacuoles was observed.…”