Early Golgi Abnormalities and Neurodegeneration upon Loss of Presynaptic Proteins Munc18-1, Syntaxin-1, or SNAP-25

Abstract

The loss of presynaptic proteins Munc18-1, syntaxin-1, or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and DRG neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 DIV upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using tetanus neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normalGolgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that DRG neurons are the only Munc18-1 KO neurons that do not degenerate or In these neurons, Golgi abnormalities were less severe, with no changes in Golgi shape. Together, these data demonstrate that cell death upon Munc18-1, syntaxin-1, or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving earlyGolgi abnormalities, distinct from apoptosis. This study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), whereas other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive cell death occurs in cultured neurons upon depleting syntaxin-1, Munc18-1, and/or SNAP-25, well before synapse formation. This study characterizes several relevant cellular phenotypes, especially early Golgi abnormalities, distinct from abnormalities observed during apoptosis, and rules out several other phenotypes as causal (defects in syntaxin-1 targeting and synaptic transmission). As proteins, such as syntaxin-1, Munc18-1, or SNAP-25, modulate α-synuclein neuropathy and/or are dysregulated in Alzheimer's disease, understanding this type of neurodegeneration may provide new links between synaptic defects and neurodegeneration in humans.

Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy

“…In these initial experiments, we used microdot cultures, where single neurons grow on islands of rat astrocytes. This reduced and standardized preparation allows quantitative comparisons between neurons expressing different variants for many morphological and functional parameters ( Wierda et al , 2007 ; Schmitz et al , 2011 ; Santos et al , 2017 ). Subsequently, we tested a selection of these variants in neurons carrying a single copy of the Stxbp1 gene to model the situation in patients (see below).…”

“…The most firmly established molecular function of Munc18-1 is to prepare synaptic vesicles for release in the presynaptic nerve terminal (reviewed in Toonen and Verhage, 2007 ). Consequently, in the absence of Munc18-1, neurons have no synaptic activity, not even spontaneous activity ( Verhage et al , 2000 ) and die within a few days in vitro and in vivo ( Verhage et al , 2000 ; Heeroma et al , 2004 ; Santos et al , 2017 ). Expression of five human variants in Stxbp1 null mutant neurons rescued neuronal viability: C180Y, M433R, G544D, T574P and C522R ( Fig.…”

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

“…Most reports that involve GA in cell death signaling are dealing with neurodegenerative diseases (Passemard et al , ). For example, cultured neurons from the central nervous system or dorsal root ganglia degenerate while manifesting early cis ‐GA abnormalities upon depletion of proteins involved in synaptic transmission (syntaxin‐1, Munc18‐1, SNAP‐25) (Santos et al , ). Similarly, specific removal of GA‐relevant proteins can precipitate neurodegeneration in mice (Liu et al , ) .…”

MUNC18–1 gene abnormalities are involved in neurodevelopmental disorders through defective cortical architecture during brain development

“…On the other hand, since it has been reported that Munc18–1 controls aggregative propensity of α-synuclein and that C180Y mutation induces abnormal aggregation of α-synuclein, the migration defects observed in this study might be attributable to the level of toxic α-synuclein aggregation [ 17 ]. In this context, disrupted function of Munc18–1 has been shown to trigger neuronal degeneration [ 53 , 54 ]. Given that clinical symptoms are different in respective patients with MUNC18–1 gene abnormalities, additional environmental or genetic factors affecting neurodegeneration are suggested in each patient.…”