Retrograde transport defects in Munc18-1 null neurons explain abnormal Golgi morphology

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its t-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether these Golgi abnormalities cause defects in the constitutive and regulated secretory pathway that may explain neurodegeneration. Electron microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. Anterograde ER-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking. We conclude that MUNC18-1 deficient neurons have normal anterograde yet reduced retrograde transport to the Golgi. This imbalance in transport routes provides a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons. Significance statement Loss of MUNC18-1 or its t-SNAREs SNAP25 and syntaxin-1 leads to massive, yet unexplained, neurodegeneration. Previous research showed that Golgi abnormalities are the earliest, shared phenotype. Golgi abnormalities are also an early feature in neurodegenerative diseases, such as Alzheimer’s Disease or Amyotrophic Lateral Sclerosis. This study elucidates the mechanism underlying the Golgi phenotype upon loss of MUNC18-1. By systematically assessing transport routes to and from the Golgi, we show that retrograde endosome-to-Golgi, but not anterograde transport from the Golgi, is disturbed. This imbalance in transport routes provides a plausible explanation for the Golgi phenotype, and may explain the neurodegeneration. The findings in this study contributes to new insights in cellular mechanisms of neurodegeneration.


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For retrograde labelling of Cholera Toxin Subunit B (CTB), neurons were incubated with 148 CTB-488 (100 ng/ml, Thermo Fisher) for 15 minutes at 37°C with 5% CO 2 in the dark. After washing 149 the cultures with warm Neurobasal, retrograde trafficking was allowed for another 2 hours before 150 neurons were fixed and stained with normal immunocytochemistry protocol.

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Images were made on a Zeiss 510 Meta Confocal microscope (Carl Zeiss) with 40X oil 152 immersion objective (NA = 1.3; Carl Zeiss). Z stacks were acquired with 0.5 or 1 μm intervals. Z 153 stacks were collapsed to maximal projections for image analysis.              intensity was ~55% lower (Fig. 4D). However, these differences between N-Cadherin and TrkB 273 staining were not specific for the Golgi marker areas. The total staining intensity in the dendrites 274 and soma was also similar for N-Cadherin, but also ~55% reduced for TrkB ( Fig. 4E-F). Hence, TrkB 275 levels are reduced in Munc18-1 KO neurons, not specifically in the Golgi and no evidence for specific 276 Golgi retention was obtained for either marker.

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To confirm this conclusion quantitatively, TrkB staining intensity in GM130-positive area was 278 divided by the staining intensity in the total neuron. For both N-Cadherin and TrkB, no differences 279 were found in the relative levels in the GM130-positive areas compared to the rest of the neuron 280 ( Fig. 4G-H). Together, these data show that although some proteins show lower expression levels, 281 no evidence was obtained for specific Golgi retention, suggesting that Golgi function is not affected 282 by its smaller size and altered shape in Munc18-1 KO neurons.         (Fig. 7A-B). Subsequently, live neurons were incubated with 343 a TrkB antibody. To distinguish surface from internalized TrkB, both fractions were stained with a 344 different secondary antibody (Fig. 7C). In both WT and Munc18-1 KO neurons a punctate pattern of internalized TrkB was observed (Fig. 7D). The mean TrkB punctum intensity in Munc18-1 KO 346 neurons was ~30% and ~35% lower in the soma and dendrites, respectively (Fig. 7E-F). Conversely, 347 the number of TrkB puncta was ~20% higher in the soma of Munc18-1 KO neurons, but not different 348 in neurites (Fig. 7G-H). Puncta size was increased by 15% and 10% in soma and dendrites, 349 respectively ( Fig. 7I-J). The total pool of internalized TrkB, measured by multiplying punctum 350 intensity by total puncta area, remained unaffected in Munc18-1 KO neurons (Fig. 7K). These data 351 together show that endocytosed TrkB puncta were bigger and contained less TrkB in Munc18-1 KO 352 neurons. Moreover, the density of TrkB puncta was higher in the soma, but not in dendrites, while 353 the total pool of internalized TrkB was unaffected. Together with the impaired retrograde CTB 354 transport, we conclude that retrograde trafficking is affected by MUNC18-1 deficiency and may 355 explain the morphological abnormalities in the Golgi in Munc18-1 KO neurons.

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In the present study we investigated the impact of MUNC18-1 loss in cellular trafficking routes. We

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but not all proteins was detected at this stage ( Fig. 4 & 5), which is a common feature in other cell 371 death models (e.g., Patel et al. 2002). Cell death in other cell types is known to also occur within 372 hours, e.g. at a rate of 5% per hour (Wolbers et al., 2004), but such high rates are induced by lethal    normalizing for the total internalized CTB pool (Fig. 6K), and in the TrkB assay the total pool of 426 internalized TrkB was unaffected (Fig. 7K). Hence, we conclude that the retrograde trafficking