Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.
The continuous release of neurotransmitter could be seen to place a persistent burden on presynaptic proteins, one that could compromise nerve terminal function. This supposition and the molecular mechanisms that might protect highly active synapses merit investigation. In hippocampal cultures from knock-out mice lacking the presynaptic cochaperone cysteine string protein-␣ (CSP-␣), we observe progressive degeneration of highly active synaptotagmin 2 (Syt2)-expressing GABAergic synapses, but surprisingly not of glutamatergic terminals. In CSP-␣ knock-out mice, synaptic degeneration of basket cell terminals occurs in vivo in the presence of normal glutamatergic synapses onto dentate gyrus granule cells. Consistent with this, in hippocampal cultures from these mice, the frequency of miniature IPSCs, caused by spontaneous GABA release, progressively declines, whereas the frequency of miniature excitatory AMPA receptormediated currents (mEPSCs), caused by spontaneous release of glutamate, is normal. However, the mEPSC amplitude progressively decreases. Remarkably, long-term block of glutamatergic transmission in cultures lacking CSP-␣ substantially rescues Syt2-expressing GABAergic synapses from neurodegeneration. These findings demonstrate that elevated neural activity increases synapse vulnerability and that CSP-␣ is essential to maintain presynaptic function under a physiologically high-activity regimen.
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