The release of neurotransmitters by Ca²⁺-evoked synaptic vesicle exocytosis is a central event in neuronal communication. This process involves a series of steps that include tethering of synaptic vesicles (SVs) to specialized areas of the plasma membrane called active zones, priming of the vesicles to a release-ready state and Ca²⁺-triggered fusion of the vesicle and plasma membranes (Südhof, 2013). Neurotransmitter release does not merely constitute a means to transmit signals between neurons. The efficiency of release is regulated by a wide variety of mechanisms during presynaptic plasticity processes that shape the properties of neural networks and underlie multiple forms of information processing in the brain (Regehr, 2012). Hence, presynaptic terminals can be viewed as minimal computational units of the brain, and understanding the mechanisms that modulate neurotransmitter release is crucial to understand brain function.

The sophisticated protein machinery that controls neurotransmitter release has been extensively characterized (Brunger et al., 2018; Rizo, 2018), yielding defined models for the functions of the central components of this machinery and allowing reconstitution of fundamental features of synaptic exocytosis in liposome fusion assays that reproduce the critical functional importance of each one of these components (Ma et al., 2013; Stepien and Rizo, 2021). The SNAP receptor (SNARE) proteins syntaxin-1, SNAP-25 and synaptobrevin play a key role in membrane fusion by forming a tight SNARE complex that consists of a four-helix bundle and brings the vesicle and plasma membranes together (Hanson et al., 1997; Poirier et al., 1998; Söllner et al., 1993; Sutton et al., 1998). N-ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the cis-SNARE complexes that result after fusion, recycling the SNAREs (Mayer et al., 1996; Söllner et al., 1993). NSF and SNAPs also dissociate trans-SNARE complexes and other four-helix bundles formed by the SNAREs (Choi et al., 2018; Ma et al., 2013; Prinslow et al., 2019; Yavuz et al., 2018), thus hindering fusion mediated by the SNAREs alone but at the same time ensuring that release occurs through a highly regulated fusion pathway that requires Munc18-1 and Munc13s (Park et al., 2017; Stepien et al., 2019), and that ensures proper parallel assembly of the SNARE four-helix bundle (Lai et al., 2017). Munc18-1 and Munc13s are essential for neurotransmitter release (Augustin et al., 1999; Richmond et al., 1999; Varoqueaux et al., 2002; Verhage et al., 2000) and organize trans-SNARE complex assembly via an NSF-SNAP-resistant pathway that starts with Munc18-1 bound to a self-inhibited ‘closed’ conformation of syntaxin-1 (Dulubova et al., 1999; Misura et al., 2000). Munc18-1 also binds to synaptobrevin, forming a template to assemble the SNARE complex (Baker et al., 2015; Jiao et al., 2018; Parisotto et al., 2014; Sitarska et al., 2017) while Munc13-1 bridges the vesicle and plasma membranes (Liu et al., 2016; Quade et al., 2019) and helps to open syntaxin-1 (Ma et al., 2011; Yang et al., 2015). Synaptic vesicle fusion upon Ca²⁺ influx is triggered by the Ca²⁺ sensor synaptotagmin-1 (Fernández-Chacón et al., 2001).

Munc13s act as master regulators of release through their multidomain architecture. Munc13-1, the major brain isoform, includes a variable N-terminal region and a C-terminal region that is conserved in all Munc13 isoforms (Figure 1A). The N-terminal region contains a calmodulin-binding region involved in Ca²⁺-dependent short-term plasticity (Junge et al., 2004) and a C₂A domain that forms a homodimer as well as a heterodimer with αRIMs, coupling RIM-dependent forms of plasticity to the priming machinery (Betz et al., 2001; Camacho et al., 2017; Deng et al., 2011; Dulubova et al., 2005; Lu et al., 2006). The conserved C-terminal region of Munc13-1 includes: (i) the C₁ domain, which mediates diacylglycerol (DAG)/phorbol ester-dependent potentiation of release (Basu et al., 2007; Rhee et al., 2002); (ii) the C₂B domain, which binds Ca²⁺ and phosphatidylinositol 4,5-bisphosphate (PIP₂) and is involved in Ca²⁺-dependent short-term plasticity (Shin et al., 2010); (iii) the MUN domain, which facilitates syntaxin-1 opening (Ma et al., 2011; Magdziarek et al., 2020); and (iv) the C₂C domain, which binds weakly to membranes in a Ca²⁺-independent manner (Quade et al., 2019). The crystal structure of a Munc13-1 fragment containing the C₁, C₂B and MUN domains (Xu et al., 2017) revealed that the C₁ and C₂B domains pack at one end of the highly elongated MUN domain, with their DAG- and Ca²⁺/PIP₂-binding sites pointing to the same direction (Figure 1A). The C₂C domain [illustrated by a homology model of the C₂C domain (Quade et al., 2019) in Figure 1A] is expected to emerge on the opposite end of the MUN domain. This architecture enables the membrane bridging activity of Munc13-1, which led to a model whereby the C₂C domain binds to the vesicle membrane while the C₁-C₂B region binds to the plasma membrane (Liu et al., 2016; Quade et al., 2019; Xu et al., 2017). This model was supported by the finding that mutations in predicted membrane-binding residues of the C₂C domain dramatically impaired the liposome clustering ability of a fragment spanning the Munc13-1 C₁, C₂B, MUN and C₂C domains (C₁C₂BMUNC₂C) in vitro, as well as vesicle docking and neurotransmitter release in neurons (Quade et al., 2019).

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The advances made in understanding the core of the release machinery provide a framework to elucidate the molecular mechanisms that underlie Munc13-1-dependent presynaptic plasticity. A model of how increases in DAG and intracellular Ca²⁺ levels during repetitive stimulation facilitate vesicle priming and neurotransmitter release emerged from the crystal structure of a Munc13-1 C₁C₂BMUN fragment and the realization that the C₁ and C₂B domains could bind to the plasma membrane through their DAG- and Ca²⁺/PIP₂-binding sites in a Ca²⁺-dependent manner, and through a polybasic region that partially overlaps with these sites in the absence of Ca²⁺ (referred to below as the polybasic face; see Figure 1B and C; Xu et al., 2017). These observations, together with liposome clustering and liposome fusion data, suggested that Munc13-1 can bridge a vesicle to the plasma membrane in two orientations (Liu et al., 2016; Quade et al., 2019; Xu et al., 2017): a close to perpendicular orientation that involves the polybasic face of the C₁-C₂B region is favored in the absence of Ca²⁺ and facilitates partial assembly of SNARE complexes but hinders progress toward membrane fusion; and a more slanted orientation that also involves the C₁-C₂B region is favored by Ca²⁺ and DAG, and stimulates further SNARE complex assembly and fusion (Figure 1D and E). Interestingly, a large amount of experimental data on short-term presynaptic plasticity accumulated over the years can be explained by a related model whereby there is a dynamic equilibrium between two primed states that involve different orientations of Munc13-1 and different extents of SNARE complex assembly, and the equilibrium can be shifted by Ca²⁺ and DAG (Neher and Brose, 2018). The proposed dual inhibitory and stimulatory roles of the membrane bridging activity of Munc13-1 are also consistent with the effects of point mutations that disrupt interactions between the various Munc13-1 domains (Xu et al., 2017), with the finding that a H567K mutation that unfolds the C₁ domain of Munc13-1 impairs vesicle priming but increases the vesicular release probability (Basu et al., 2007; Rhee et al., 2002), and with the observation that deletion of the C₁ or C₂B domain of unc-13 enhances neurotransmitter release in C. elegans, yet deletion of both domains strongly hinders release (Michelassi et al., 2017). Nevertheless, the validity of this two-state model and the functional importance of the polybasic face formed by the C₁ and C₂B domains remain to be demonstrated.

The study presented here was designed to investigate the functional consequences of mutations in predicted membrane-binding residues of the Munc13-1 C₁-C₂B region and test this model using a combination of molecular dynamics (MD) simulations, biochemical and reconstitution assays in vitro, and electrophysiological experiments in neurons. We find that K603E or R769E single point mutations in the C₁C₂B polybasic face of Munc13-1 disrupt Ca²⁺-independent liposome binding and bridging, as well as stimulation of liposome fusion in reconstitution assays, and severely impair synaptic vesicle priming in mice autaptic cultures. Conversely, a K720E mutation in the polybasic face and a K706E mutation in the C₂B domain Ca²⁺-binding loops, which have milder effects on liposome binding, bridging and fusion, do not affect vesicle priming; however, Ca²⁺-evoked release and the release probability are enhanced by the K720E mutation and impaired by K706E. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. These results strongly support the notion that binding of the C₁-C₂B region of Munc13-1 to the plasma membrane is critical for synaptic vesicle priming and that two distinct membrane-binding faces of this region control neurotransmitter release.

Research for over three decades has led to major advances in our understanding of the basic mechanisms underlying the priming and fusion of synaptic vesicles to release neurotransmitters, including the notions that Munc18-1 and Munc13-1 coordinate assembly of SNARE complexes in an NSF-αSNAP-resistant manner (Ma et al., 2013; Prinslow et al., 2019) and that this pathway enables the multiple modes of regulation of release probability during presynaptic plasticity that depend on Munc13-1 (Park et al., 2017; Sitarska et al., 2017; Stepien et al., 2019). This large multiple domain protein plays fundamental roles in this process by accelerating opening of the syntaxin-1 closed conformation (Ma et al., 2011; Yang et al., 2015) and bridging the vesicle and plasma membranes (Liu et al., 2016; Quade et al., 2019). Thus, it seems likely that Munc13-1-dependent regulation of neurotransmitter release involves at least in part alteration of these activities. Indeed, the finding that the C₁C₂B region has two putative faces that can bind to membranes led to an intriguing model postulating that membrane bridging by Munc13-1 in two orientations modulates neurotransmitter release (Xu et al., 2017). Here, we have tested this model and examined the role of the C₁C₂B region in neurotransmitter release. We find that basic residues in this region play key roles in SV priming and in dictating the vesicular release probability, and also modulate short-term plasticity. The finding that two faces of the Munc13-1 C₁C₂B region play important roles in neurotransmitter release support the model of Figure 1D and E, but other interpretations are possible.

The notion that the highly conserved C-terminal region of Munc13-1 containing the C₁, C₂B, MUN and C₂C domains bridges the vesicles and plasma membranes arose naturally from the discovery of its rod-like architecture, with membrane binding domains on opposite ends of the highly elongated MUN domain (Liu et al., 2016; Xu et al., 2017). At the same time, the structural studies revealed that the Munc13-1 C₁C₂B region contains a large polybasic face adjacent to the region containing the DAG/phorbol ester-binding site of the C₁ domain and the Ca²⁺/PIP₂-binding region of the C₂B domain (Figure 1B and C), suggesting that Munc13-1 can bind to the plasma membrane through two different faces that yield different orientations (Xu et al., 2017). Although our MD simulations need to be interpreted with caution because of the limited simulation times, they yield binding modes that make sense from a biophysical point of view and indicate that membrane interactions mediated by the polybasic face involve multiple ionic interactions in an extensive surface, leading to an almost perpendicular orientation of the Munc13-1 rod with respect to the membrane. In contrast, the simulations suggest that interactions mediated by the DAG/Ca²⁺/PIP₂-binding face involve a smaller area and are favored by Ca²⁺-phospholipid coordination and insertion of hydrophobic residues into the membrane in addition to ionic interactions, leading to a very slanted orientation (Figure 1D and E, Figure 1—figure supplements 1 and 2). The relevance of the two binding modes observed in the simulations is supported by the biochemical and physiological effects of the mutations described here and those caused by mutations that disrupt DAG/phorbol ester binding to the C₁ domain (Rhee et al., 2002) or Ca²⁺ binding to the C₂B domain (Shin et al., 2010). We also note that the structure of the C₁C₂BMUN fragment appears to be rather rigid (Xu et al., 2017) and there is no apparent reason why Ca²⁺ binding to the C₂B domain should alter the overall structure of the Munc13-1 C-terminal region. Hence, although our data do not demonstrate that two orientations of Munc13-1 with respect to the plasma membrane are physiologically relevant, this notion is supported by the functional importance of the two binding faces, as they are expected to lead to two drastically different orientations.

It is important to realize that the orientation of Munc13-1 is likely dynamic, particularly in the presence of other forces. For instance, the perpendicular orientation of Munc13-1 may facilitate initiation of SNARE core complex assembly but prevent full assembly, resulting in a tug-of-war between Munc13-1 and the SNAREs in the absence of Ca²⁺ that is dramatically tilted toward full SNARE complex assembly and fusion when Ca²⁺ binding to the C₂B domain favors slanted orientations. However, the balance can also be tilted towards slanted orientations in the absence of Ca²⁺ by the energy associated with further assembly of the SNARE complex and by proteins that bind to the SNARE complex such as Syt1 and complexins (see below). Indeed, a large amount of physiological data can be explained with a model postulating that primed vesicles exist in a dynamic equilibrium between a loosely primed state (LS), in which SNARE complexes are partially assembled and Munc13-1 bridges the vesicle and plasma membrane in a perpendicular orientation, and a tightly primed state (TS) where the SNARE complex is more fully assembled and Munc13-1 bridges the membranes in a slanted orientation (Neher and Brose, 2018). In this scenario, vesicles in the TS state are much more likely to be released by an action potential than those in the LS state, and accumulation of Ca²⁺ during repetitive stimulation increases the vesicular release probability because it shifts the equilibrium toward the TS state. Note, however, that methods used to measure the RRP, such as application of sucrose, likely release vesicles in both the LS and TS states. This two-state model and the proposed nature of the two membrane-binding modes of the C₁-C₂B region underlying the two states are supported by our data in combination with previous studies (Rhee et al., 2002; Shin et al., 2010).

The strong impairments of liposome binding and clustering in the absence of Ca²⁺ caused by the K603E and R769E mutations in Munc13-1 C₁C₂BMUNC₂C (Figures 2 and 3) show that the polybasic face is indeed critical for Ca²⁺-independent membrane binding and membrane bridging. These mutations also caused the strongest impairments of Ca²⁺-independent liposome fusion among the single mutants (Figure 5C, D and F). The finding that the K720E mutation in the polybasic face has smaller effects on Ca²⁺-independent liposome binding, clustering and fusion suggests that K720 has a smaller energetic contribution to membrane binding than those of K603 and R769. The K706E mutation exhibited the smallest inhibitory effects on liposome binding and fusion in the absence of Ca²⁺, which correlates with the fact that K706 is not in the polybasic face.

Ca²⁺ enhanced the affinity of all the mutants for liposomes (Figure 2) and increased the clustering activity of the mutants that had the weakest clustering ability (K603E, R769E, and the two double mutants), except the quadruple mutant (Figure 3). Hence, although we did not observe Ca²⁺-induced increases in liposome binding and clustering for WT C₁C₂BMUNC₂C because its liposome affinity and clustering activity are very high, Ca²⁺ did increase binding and clustering when these activities were impaired by mutation. These increases may arise in part because Ca²⁺ binding to the C₂B domain enhances the overall positive electrostatic potential and in part because the Ca²⁺-dependent binding mode with slanted orientations occurs with a higher affinity than the Ca²⁺-independent binding mode involving the polybasic face. The four single mutants and the K603E/K720E double mutant supported Ca²⁺-dependent fusion with comparable efficiency as WT C₁C₂BMUNC₂C (Figure 5A, B and E) even though the Ca²⁺-dependent clustering activity of all these mutants was somewhat lower than that of WT (Figure 3), suggesting that fusion efficiency can remain very high as long as the clustering activity is above a certain threshold. Interestingly, the double K706E/R769E mutation in the C₂B domain Ca²⁺-binding sites disrupted Ca²⁺-dependent liposome fusion severely (Figure 5A, B and E) even though its clustering activity is comparable to that of C1C2BMUNC2C with the K603E/K720E double mutation in the polybasic face (Figure 3). This finding strongly supports the notion that a Ca²⁺-induced switch to a slanted orientation is critical for Ca²⁺-induced enhancement of liposome fusion.

Our electrophysiological studies support the biological relevance of at least some of the results obtained in our in vitro assays and reveal additional effects of the mutations that could not be discerned with the liposome fusion assays, providing critical evidence supporting the functional relevance of the two faces of the Munc13-1 C₁C₂B region. The observation that only the K603E and R769E mutants among the single Munc13-1 mutants exhibited severely impaired SV priming (Figure 6A) clearly correlates with the much stronger disruption of Ca²⁺-independent liposome binding, clustering and fusion caused by these mutations, compared to the K706E and K720E single mutants (Figures 2, 3, 5C, D and F). These results provide compelling evidence that the polybasic face of the Munc13-1 C₁C₂B region plays a critical function in synaptic vesicle priming. Together with the key importance of the C₂C domain for membrane bridging and SV priming (Quade et al., 2019), these data strongly support the notion that this critical function involves bridging of the vesicle and plasma membranes by respective interactions involving the C₂C domain and the C₁C₂B region of Munc13-1. The K603E and R769E mutants also exhibited similar impairments in Ca²⁺-evoked release that arose because of the defects in priming, as the Pvr remained analogous to that of WT Munc13-1 (Figure 6B–C), and the potentiation of release by PDBu was somewhat larger for both mutants than for WT Munc13-1 (Figure 10A). These observations suggest that binding of PDBu to the C₁ domain can partially compensate for the defects in priming caused by these two mutations by favoring the binding mode involving the slanted orientation, which increases vesicle fusogenicity. Interestingly, we observed a considerably stronger depression during a 10 Hz stimulus train for the R769E mutant than for the K603E mutant (Figure 8). This finding supports the notion that accumulation of Ca²⁺ during repetitive stimulation facilitates the transition to slanted orientations, which are expected to be destabilized by the R769E mutation but not by the K603E mutation.

It was surprising that the K720E mutation in the polybasic interface did not impair SV priming and enhanced Ca²⁺-evoked release as well as the Pvr, in contrast to the effects of the K603E in a nearby basic residue (Figure 6A–C). However, as mentioned above, there is likely a delicate balance between inhibitory and stimulatory interactions within the release machinery, and some of the interactions involving the polybasic region that are important for priming initiated by a perpendicular orientation of Munc13-1 need to be released to transit to slanted orientations that support full zippering of the SNARE complex and membrane fusion. Since the K720E mutation has only a modest effect on Ca²⁺-independent liposome binding and clustering (Figures 2 and 3D), a likely explanation of our results is that the mutant retains sufficient membrane affinity to fully support priming and facilitates the transition to the more active slanted orientations, yielding a higher release probability. This model is consistent with the finding that, in contrast to the K603E mutant, the K720E mutant exhibited less potentiation of release by PDBu than WT (Figure 10A), likely because this mutation already promotes the slanted orientations that are favored by PDBu binding to the C₁ domain. In 10 Hz stimulus trains, the K720E mutant depressed initially more than WT Munc13-1, but later exhibited analogous EPSC amplitudes as WT (Figure 8D–F), likely because accumulation of Ca²⁺ during repetitive stimulation already induces slanted orientations that do not involve interactions of K720 with the membrane. Nevertheless, we cannot rule out the possibility that the effects of the K720E mutation arise from a different mechanism, for instance if the mutation alters an as yet unidentified interaction of Munc13-1.

The K706E mutation did not affect SV priming, in agreement with the notion that K706 does not participate in the interactions of the polybasic interface that mediate priming, but this mutant did decrease Ca²⁺-evoked release and hence the probability of release (Figure 6A–C). These effects are opposite to those caused by mutation of the corresponding lysine residue of ubMunc13-2 to Trp (the KW mutant) (Shin et al., 2010), which caused a gain-of-function, and could arise in principle because the K706E mutation hinders the transition to slanted orientations. This interpretation is consistent with the observation that EPSCs remained lower than WT for the K706E mutant during 10 Hz repetitive stimulation (Figure 8G–I). However, the observation that PDBu potentiation was similar for this mutant and WT Munc13-1 (Figure 10A) suggests that the effects of the K706E mutation might not be related to the transition to slanted orientations but rather to another mechanism that directly influences fusion. For instance, the Munc13-1 C₂B domain might cause membrane perturbations analogous to those that are believed to underlie the function of the Syt1 C₂ domains in triggering release (Fernández-Chacón et al., 2001; Rhee et al., 2005). It is also possible that the phenotypes caused by the K706E mutation and other mutations studied here reflect effects of Munc13-1 in more than one step leading to release, which complicates the interpretation of the data.

Despite these uncertainties in the interpretation of our results, the overall data strongly support the notions that the Munc13-1 C₁C₂B region plays a critical role in SV priming and that this region underlies two types of interactions with the plasma membrane that yield different orientations of Munc13-1. This model is also consistent with the previous observation that a mutation that is expected to unfold the Munc13-1 C₁ domain leads to decreased priming but increased release probability in mice (Basu et al., 2007; Rhee et al., 2002), as the absence of the C₁ domain is expected to impair the initial binding to the plasma membrane that is important for priming but likely helps to adopt slanted orientations that facilitate release. Similarly, the finding that deletion of the C₁ domain or the C₂B domain of C. elegans unc-13 enhances release but deletion of both domains strongly impairs release (Michelassi et al., 2017) suggest that both the C₁ domain and the C₂B domain are important to stabilize the perpendicular orientations that mediate priming but hinder transition to the active, slanted orientations; however, at least one of the two domains needs to be present to mediate the interaction with the plasma membrane.

Further research will be necessary to better understand the nature of the LS and TS states and the factors that influence the postulated equilibrium between them. Thus, the equilibrium is expected to be affected by Syt1 and complexins, which are believed to maintain trans-SNARE complexes in a state that is ready for fast release but is inhibited to prevent premature fusion (Voleti et al., 2020), and that most likely corresponds to the TS state. Note that the release probability and the RRP are reduced in Syt1 KO mice (Chang et al., 2018) while, in the absence of complexins, the release probability is also decreased but not the RRP (Reim et al., 2001). Since Syt1 promotes trans-SNARE complex assembly and complexins protect trans-SNARE complexes against disassembly by NSF/αSNAP (Prinslow et al., 2019), stabilizing the C-terminus of the SNARE four-helix bundle (Chen et al., 2002), it is tempting to speculate that Syt1 facilitates initial formation of LS and complexins shift the equilibrium toward TS. Other proteins such as CAPS, which has functions related to those of Munc13s (Jockusch et al., 2007), may also affect the equilibrium between the two primed states. Clearly, much we have learned but much remains to be learned about this fascinating system.

Source data files are provided for all data figure panels.

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Author details

1. Marcial Camacho

   1. Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Neurophysiology, Berlin, Germany
   2. NeuroCure Cluster of Excellence, Berlin, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft

   Contributed equally with

   Bradley Quade

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2367-1259

2. Bradley Quade

   1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
   3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - original draft

   Contributed equally with

   Marcial Camacho

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5330-1355

3. Thorsten Trimbuch

   1. Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Neurophysiology, Berlin, Germany
   2. NeuroCure Cluster of Excellence, Berlin, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Junjie Xu

   1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
   3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Levent Sari

   1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
   2. Cecil H. and Ida Green Comprehensive Center for Molecular, Computational and Systems Biology, University of Texas Southwestern Medical Center, Dallas, United States
   3. Center for Alzheimer’s and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Josep Rizo

   1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
   3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft

   For correspondence

   Jose.Rizo-Rey@UTSouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1773-8311

7. Christian Rosenmund

   1. Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Neurophysiology, Berlin, Germany
   2. NeuroCure Cluster of Excellence, Berlin, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft

   For correspondence

   christian.rosenmund@charite.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3905-2444

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