STXBP1 is Critical For Normal Neuronal Development

Asish Madarapu

introduction

Neuronal communication via synapses is a vital process and a complex protein machinery maintains this process of synaptic transmission. Syntaxin binding protein 1 (STXBP1, aka, MUNC-18-1) is a presynaptic protein encoded by the STXBP1 gene .  STXBP1 is a vital part of a multi-protein structure known as the SNARE complex, which controls the fusion of synaptic vesicles to the cell membrane. Membrane fusion is a universal process in all eukaryotic cells. In the brain, synaptic vesicles fuse with the outer membrane of neurons in order to release neurotransmitters.

This essential function and role of STXBP1 in synapse function and the communication between neurons is well known and is understood to underlie the pathology associated with STXBP1-related disorders.  However, two recent papers from the Verhage lab in Amsterdam suggest that the protein may also be essential for proper synapse formation and normal neuronal development in the developing brain.

deleting stxbp1 causes neurodegeneration

A paper by Van Berkel (Ref 1) looks at which cellular processes are affected in the absence of STXBP1 in the developing mouse brain. Removal of both Stxbp1 genes in a mouse results in a Stxbp1 knock-out (KO) animal. The brain of Stxbp1 KO mice begin to develop normally but at one point, just before birth, there is substantial neurodegeneration, i.e. the neurons which had been developing normally begin to die off.  Interestingly, this occurs before the synapses are formed in the developing brain (synapses are one of the last neuronal structures to form). Additionally, it was observed that depletion of other proteins (MUNC13 and VAMP2) that play a role in synaptic transmission, does not result in neurodegeneration. This paper investigated why STXBP1 is special and how its removal causes neurodegeneration.

To see the effects of Stxbp1 deletion, the lab examined changes in the transcriptome and proteome in neurons from Stxbp1 KO neurons compared to neurons from normal mice. The transcriptome refers to all the different RNAs present in a cell or tissue. This tells you what genes are active and not active (recall that transcription is the process where cells make an RNA copy of DNA). The proteome refers to all the different proteins in a cell or tissue and tells you about how the cell or tissue is functioning. Comparison from brain tissue, removed at the time just before neurodegeneration is known to begin, demonstrated that the transcriptome of Stxbp1 KO mice was significantly different compared to normal mice. RNA sequencing showed that 11.5% of transcripts were dysregulated significantly.  The most significant changes observed  were transcripts involved in synapse function, which were significantly decreased in the brain tissue. In addition, transcripts involved in immune response and lipid metabolism were increased. Synapse function is specific to neurons, while lipid metabolism and immune response are specific to glial cells, which are non-neuronal cells in the brain that provide support to maintain neurons. This shows that loss of STXBP1 affects cells differently in the brain and may lead to neurodegeneration.

To examine specific changes in the proteome, neuronal cultures from the brains of wild type and Stxbp1 KO were used. Proteomic analysis was done twice in order to see when cells became dependent on STXBP1. In the first time point (referred to as DIV2 in the paper), 5% of the 3100 unique proteins detected were dysregulated. It rose to 13% at the second time point (DIV3). So over time, more proteins related to neuron development and synaptic function were affected and more became dysfunctional. Overall, the Stxbp1 KO neurons undergo much more remodeling than the wild type neurons, further showing the effects of depletion.

In addition to changes in the proteome, there was significant cell death between the two time periods. There are two well known types of processes associated with neuronal cell death, apoptosis and neuronal excitotoxicity; both of these processes naturally occur in the brain. To see if there was overlap, proteins regulated in the Stxbp1 KO neurons were compared with proteins typically affected during these types of cell death. There was minimal overlap with apoptosis and excitotoxicity related proteins (Figure 1 A&B). Further examination of known proteins associated with any type of cell death also showed little overlap (Figure 1 C).  This suggests that the process responsible for the neurodegeneration observed in the Stxbp1 KO mice is unique. Given the rapid cell death observed in the KO mice, STXBP1 might serve as a “checkpoint” for neuronal development. For neurons to continue to the next stage of development, the presence of STXBP1 could be required, and without it, could trigger cell death. Exactly how STXBP1 would function as a checkpoint remains to be further studied.

stxbp1 is important for both exocytosis and endocytosis

In the second paper from the Verhage lab, Lammertse addressed the question of whether STXBP1, when depleted, affects endocytic proteins (Ref 2). Endocytosis and exocytosis are both processes that involve cells moving materials in and out of them. In endocytosis, cells take substances from the outside environment and engulf them. In exocytosis, substances are pushed outside the cell. In neuronal cells, neurotransmitters and hormones are released via fusion of vesicles through exocytosis. As described in the previous paper, absence of STXBP1 protein stops exocytosis (synaptic communication) and triggers widespread neurodegeneration.

This paper demonstrates that both the RNA and protein for Dynamin-1 is reduced in Stxbp1 KO mice. Dynamin-1 is a protein important in regulating endocytosis, thus STXBP1, a protein generally associated with exocytosis appears to control the amount of Dynamin-1, a protein involved in endocytosis, in the developing brain. Given this finding, the opposite was also tested: whether absence of Dynamin-1 leads to reduction of STXBP1. This was not the case. Additionally, knocking out exocytosis-related genes other than STXBP1 (SNAP25 and MUNC13)  did not alter Dynamin-1 RNA or protein, thus the effect of STXBP1 depletion appears to be specific and unique. The reduction in Dynamin-1 in the KO mice, however, does not appear to play a role in the neurodegeneration observed in these mice as restoring the normal amount of Dynamin-1 in the Stxbp1 KO mouse neurons did not improve their ability to survive.

Taken together, these papers show how essential the presence of STXBP1 is in the developing brain and may suggest a further role for STXBP1 in STXBP1 related disorders.   Developmental delay is common amongst all STXBP1 patients. The presumed mechanism underlying this is the reduction in neuron communication due to decreases in neurotransmitter release via exocytosis. There is no neurodegeneration observed in patients but it is possible that partial loss of STXBP1 levels have an effect on neuronal and synaptic development that is yet unappreciated. This might be expected to produce a milder phenotype than that observed in KO neurons but still contribute to the pathobiology of the disorder.

References

1.   Berkel AAV., Koopmans F, Gonzalez-Lozano MA, Lammertse HCA, Feringa F, Bryois J, Sullivan PF, Smit AB, Toonen RF, & Verhage M. (2022). Dysregulation of synaptic and developmental transcriptomic/proteomic profiles upon depletion of MUNC18-1. eNeuro, 9(6):ENEURO.0186-22.2022. (pubmed: 36257704)

2. Lammertse HCA, Moro A, Saarloos I, Toonen RF, Verhage M. (2022). Reduced dynamin-1 levels in neurons lacking MUNC18-1. J. Cell Sci., 135(22)jcs260132. (pubmed: 36245272)