To achieve maturation, autophagic and endocytic vesicles undergo long-distance transport from distal regions back to the soma, a distance that can span up to 1 m long in peripheral neurons ( Overly and Hollenbeck, 1996). These unique features add yet another layer of complexity to the distribution and maturation of lysosomes. Neurons are highly polarized and postmitotic cells with three functionally and structurally different compartments: soma, dendrites, and axons. A mature lysosome is thus defined as: (A) a storage organelle for active forms of degradative enzymes with acidic pH optimum (B) the hydrolysis of substrates occurring within (C) limiting membranes with specific glycosylated membrane-associated proteins such as LAMP1 and (D) the lack of nonlysosomal proteins such as mannose-6-phosphate receptors (MPRs Saftig, 2005 Luzio et al., 2007). These heterogeneous intermediates, having diverse qualities in morphology, membrane components, hydrolase contents, luminal pH, and distinct cellular functions, represent the different stages of maturation within the endolysosomal pathway ( Saftig and Klumperman, 2009). Before endocytosed/autophagic organelles mature into lysosomes, they are a continuum of intermediates that continually pass over endocytosed cargos, exchange membrane constituents, and add lysosomal hydrolases. Autophagosomes undergo a stepwise maturation through fusion of autophagosomes with endosomes to form amphisomes or with lysosomes to form degradative autolysosomes ( Klionsky and Emr, 2000 Levine and Klionsky, 2004 Maday et al., 2012, 2014 Nixon, 2013 Cheng et al., 2015). Newly synthesized precursors of lysosomal hydrolases are transported from the TGN to endosomes and lysosomes ( Geuze et al., 1985 Griffiths et al., 1988, 1990 Lobel et al., 1989 Ishidoh and Kominami, 2002). Endolysosomal trafficking from early endosomes (EEs) to late endosomes (LEs) and finally into mature lysosomes is essential for delivering target materials and maintaining efficient degradation capacity. In addition, lysosomes play a critical role in nutrient sensing and recycling of the catabolic pathway ( Luzio et al., 2007). Lysosomal hydrolases degrade extracellular materials that have been internalized by endocytosis and intracellular components sequestered by autophagy. Lysosomes serve as the terminal degradation hubs for autophagic/endocytic components, thus maintaining cellular homeostasis essential for neuronal growth and survival as well as synaptic remodeling ( Cai et al., 2010 Farías et al., 2017 Padamsey et al., 2017). Our study suggests that labeling a set of lysosomal hydrolases combined with various endolysosomal markers would be more accurate than simply relying on LAMP1/2 staining to assess neuronal lysosome distribution, trafficking, and functionality under physiological and pathological conditions. We further reveal that LAMP1 intensity in familial amyotrophic lateral sclerosis–linked motor neurons does not necessarily reflect lysosomal deficits in vivo. Gradient fractionation displays differential distribution patterns of LAMP1/2 and cathepsins D/B in neurons. A bovine serum albumin–gold pulse–chase assay followed by ultrastructural analysis suggests a heterogeneity of degradative capacity in LAMP1-labeled endolysosomal organelles. Our study demonstrates that a significant portion of LAMP1-labeled organelles do not contain detectable lysosomal hydrolases including cathepsins D and B and glucocerebrosidase. In this study, we use immunoelectron microscopy and confocal imaging to provide quantitative analysis of LAMP1 distribution in various autophagic and endolysosomal organelles in neurons.

Despite widespread distribution of LAMP1 and the heterogeneous nature of LAMP1-labeled compartments, LAMP1 is routinely used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomes.

Dendrite labeled