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After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there.

# These authors contributed equally to this work preventing high-molecular weight molecules from passing through [25]. Indeed, the design and engineering of NP with high specificity for brain capillary endothelial cells have been proposed as promising strategy for AD diagnosis and treatment [26][27][28][29].The aim of the present investigation was to design NP able to bind Aβ peptide and to cross the BBB. To reach this goal we developed nanoliposomes (NL) double-functionalized with a curcuminderivative and with a modified HIV Transactivating Transcriptional Activator (TAT) peptide. Preparation of NL covalently decorated with curcumin-derivative was previously carried out, starting from a curcumin alkyne-derivative showing a very high affinity for Aβ peptide [30], suitable for NL decoration by click chemistry and with improved features of stability with respect to curcumin itself [31]. On the other side, TAT-peptide could enhance NP BBB crossing [32,33] based on the evidence that its coupling to NP may facilitate their efficient translocation through the cell membrane, bypassing the endocytic pathway [34][35][36]. TAT-peptide was covalently attached to NL surface via a thiol-maleimide reaction. The ability of NL to bind Aβ after AbstractProduction of abnormally high amounts of amyloid-β peptide in the brain plays a central role in the onset and development of Alzheimer's disease, a neurodegenerative disorder affecting millions of individuals worldwide. Nanoparticles have been proposed as promising tools to treat the disease by delivering drugs and contrast agents to the brain. Here, nanoliposomes decorated with a curcumin-derivative, displaying high affinity for amyloid-β, were functionalized with a modified cell-penetrating TAT-peptide, with the aim of conferring on such nanoliposomes the ability to cross the blood-brain barrier. Functionalization with TAT-peptide did not modify the ability of curcumindecorated nanoliposomes to bind amyloid-β fibrils, as assessed by surface plasmon resonance. Confocal microscopy, mass spectrometry and radioactivity experiments with [3 H]-sphingomyelin showed about 3-fold increase in the uptake of nanoliposomes by human brain capillary endothelial cells (hCMEC/D3) after the functionalization with TATpeptide, with no alterations in cell viability. Moreover, TAT functionalization increased the permeability of curcuminnanoliposomes across a blood-brain barrier model made with the same cells. The similar permeabilities of curcuminderivative and [3 H]-sphingomyelin suggested that nanoliposomes were transported intact. Considering these results, nanoliposomes functionalized with the curcumin-derivative and TAT-peptide represent a promising tool for targeting amyloid-β directly in the brain parenchyma.