Cell surface proteolysis is essential for communication between cells and results in the shedding of membraneprotein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizureprotein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP-43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule-associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 overexpression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant-negative Rab7-interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 + inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS-and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Doublehomozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.neurodegeneration | zinc finger nuclease | proteomics A myotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin and TDP-43 + inclusions (FTLD-TDP) are incurable fatal neurodegenerative diseases. ALS is characterized by the loss of upper and lower motor neurons and FTLD patients suffer from degeneration of the frontal and temporal lobes. Both diseases belong to an ALS-FTLD disease spectrum (1, 2). TDP-43 is a RNA binding protein, which modulates RNA splicing, and systematic cross-linking and immunoprecipitation studies identified numerous RNA targets (3-5). About 90% of ALS cases are sporadic and the majority is pathologically characterized by insoluble TDP-43 inclusions (1, 6). Mutations in TDP-43 can lead to familial forms of ALS and FTLD-TDP (7,8). Most of the patientassociated mutations cluster in the C-terminal glycine-rich domain of TDP-43 (9), which mediates protein-protein interactions and is required for splicing-associated activities as well as autoregulation (10)(11)(12)(13)(14). Pathologically, ALS and FTLD-TDP are characterized by nuclear clearance and deposition of insoluble . Whether neurotoxicity of the TDP-43 inclusions or reduced TDP-43 function upon nuclear clearance is responsible for ALS and FTLD is under debate. However, little is known about the physiological function of TDP-43. To obtain insights into the in vivo function of TDP-43, we investigated the morphological, developmental, and molecular consequences of a loss of TDP-43 in zebrafish.

Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1؊/؊ and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1؊/؊ and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1؊/؊ mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken Cerebrospinal fluid (CSF) 1 consists of interstitial fluid that is in continuous exchange with the central nervous system and the peripheral blood system. It represents the only body fluid in humans that is in direct contact with brain tissue and accessible in a routine clinical setting. Thus, the easy accessibility from the periphery renders CSF perfectly suited to study pathologic neurological processes (1). Human CSF has a relatively low protein content (ϳ 0.4 mg/ml), but features a highly diverse proteome. It is thus increasingly studied by modern mass spectrometry based proteomics (2). The proteomic analysis of human CSF typically involves various protein concentration and fractionation steps as well as the depletion of highly abundant proteins, such as serum albumin. This allows the identification of several hundred up to 2600 proteins from several milliliters of human CSF (3).Mice are the most popular animal model in preclinical research, because of their similarity to humans in genetics and physiology, their unlimited supply and their ease of genetic engineering. The study of their CSF can provide valuable insights into disease mechanisms and biomarker discovery Author Contributions: The manuscript was written by Bastian Dislich and Stefan F. Lichtenthaler. The study was guided and planned by Bastian Dislich, Sebastian Hogl, Melanie Meyer-Luehmann and Stefan F. Lichtenthaler. Bastian Dislich established the mass spectrometry of murine CSF and performed the proteomic analysis of the BACE1Ϫ/Ϫ CSF. Felix Wohlrab performed all other experiments, except for the CSF extraction, which was performed by Teresa Bachhuber, and the analysis of...