Intracellular protein-protein interactions are dynamic events requiring tightly regulated spatial and temporal checkpoints. But how are these spatial and temporal cues integrated to produce highly specific molecular response patterns? A helpful analogy to this process is that of a cellular map, one based on the fleeting localization and activity of various coordinating proteins that direct a wide array of interactions between key molecules. One such protein, myristoylated alanine-rich C-kinase substrate (MARCKS) has recently emerged as an important component of this cellular map, governing a wide variety of protein interactions in every cell type within the brain. In addition to its well-documented interactions with the actin cytoskeleton, MARCKS has been found to interact with a number of other proteins involved in processes ranging from intracellular signaling to process outgrowth. Here, we will explore these diverse interactions and their role in an array of brain-specific functions that have important implications for many neurological conditions.

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Introduction: CLN3 Batten disease is a rare pediatric neurodegenerative lysosomal disorder caused by biallelic disease-associated variants in CLN3. Despite decades of intense research, specific biofluid biomarkers of disease status have not been reported, hindering clinical development of therapies. Thus, we sought to determine whether individuals with CLN3 Batten disease have elevated levels of specific metabolites in blood. Methods: We performed an exhaustive metabolomic screen using serum samples from a novel minipig model of CLN3 Batten disease and validated findings in CLN3 pig serum and CSF and Cln3 mouse serum. We further validate biomarker candidates with a retrospective analysis of plasma and CSF samples collected from participants in a natural history study. Plasma samples were evaluated from 22 phenotyped individuals with CLN3 disease, 15 heterozygous carriers, and 6 non-affected non-carriers (NANC). Results: CLN3 pig serum samples from 4 ages exhibited large elevations in 4 glycerophosphodiester species: glycerophosphoinositol (GPI), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), and glycerophosphoserine (GPS). GPI and GPE exhibited the largest elevations, with similar elevations found in CLN3 pig CSF and Cln3 mouse serum. In plasma samples from individuals with CLN3 disease, glycerophosphoethanolamine and glycerophosphoinositol were significantly elevated with glycerophosphoinositol exhibiting the clearest separation (mean 0.1338 vs 0.04401 nmol/mL for non-affected non-carriers). Glycerophosphoinositol demonstrated excellent sensitivity and specificity as a biomarker, with a receiver operating characteristic area under the curve of 0.9848 ( P = .0003). Conclusions: GPE and GPI could have utility as biomarkers of CLN3 disease status. GPI, in particular, shows consistent elevations across a diverse cohort of individuals with CLN3. This raises the potential to use these biomarkers as a blood-based diagnostic test or as an efficacy measure for disease-modifying therapies.

Axons of the corpus callosum (CC), the white matter tract that connects the left and right hemispheres of the brain, receive instruction from a number of chemoattractant and chemorepulsant cues during their initial navigation towards and across the midline. While it has long been known that the CC is malformed in the absence of Myristoylated alanine-rich C-kinase substrate (MARCKS), evidence for a direct role of MARCKS in axon navigation has been lacking. Here, we show that MARCKS is necessary for Netrin-1 (NTN1) signaling through the DCC receptor, which is critical for axon guidance decisions. Marcks null (Marcks) neurons fail to respond to exogenous NTN1 and are deficient in markers of DCC activation. Without MARCKS, the subcellular distributions of two critical mediators of NTN1-DCC signaling, the tyrosine kinases PTK2 and SRC, are disrupted. Together, this work establishes a novel role for MARCKS in axon dynamics and highlights the necessity of MARCKS as an organizer of DCC signaling at the membrane.

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo , thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

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Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo, demonstrating a novel shared etiology.