Sanfilippo syndrome is an inherited lysosomal disease associated to defective autophagy-lysosomal pathway (ALP). Neuropathy is a predominant feature of Sanfilippo patients, however, to date, it is still uncurable. Recently, we found that Sanfilippo disease is characterized by a progressive aggregation of amyloid proteins (mostly α-synuclein), which affect ALP, thus generating a neurotoxic vicious cycle. Nevertheless, the mechanisms by which amyloid aggregation causes ALP dysfunction remains partially elucidated. By using Sanfilippo mouse brain samples and primary neuronal cultures, we have demonstrated that amyloid proteins accumulate in the proximity of lysosomes, where they cause massive lysosomal enlargement and perinuclear clustering. This, in turns, impairs the lysosomal capability to encounter and clear autophagosomes to complete autophagy. Nuclear magnetic resonance data and experiments in a cell-free system, showed that such effect is mediated by the direct binding of α-synuclein to the lysosomal membrane that induces the assembly and fusion of lysosomes in large structures through the ability of α- synuclein to form a "double-anchor" between lysosomes and to self-aggregate. It is known that lysosomal size and dynamic is functionally associated to the Endoplasmic Reticulum (ER) membrane organization and, specifically, to the ER-lysosome contact sites. Therefore, we hypothesize that the amyloid-mediated increase in the lysosomal size may alter ER-lysosome contact sites, thus reducing lysosomal dynamics and availability for proper neuronal function, including autophagy. Our results uncover a new mode of action by which amyloid deposition affect lysosomal function, thus identifying alternative druggable mechanisms, which may be relevant not only for the treatment of Sanfilippo disease but also for other neurodegenerative conditions associated to amyloid aggregation and ALP dysfunction.