Pannexin 1 regulates spiny protrusion dynamics in cortical neurons

The integration of neurons into networks relies on the formation of dendritic spines. These specialized structures arise from dynamic filopodia-like spiny protrusions. Recently, it was discovered that cortical neurons lacking the channel protein Pannexin 1 (Panx1) exhibited larger and more complicated neuronal networks, as well as, higher dendritic spine densities. Here, we expanded on those findings to investigate whether the increase in dendritic spine density associated with lack of Panx1 was due to differences in the rates of spine dynamics. Using a fluorescent membrane tag (mCherry-CD9-10) to visualize spiny protrusions in developing neurons (at 10 days-in-vitro, DIV10) we confirmed that lack of Panx1 leads to higher spiny protrusion density while transient transfection of Panx1 leads to decreased spiny protrusion density. To quantify the impact of Panx1 expression on spiny protrusion formation, elimination, and motility, we used live cell imaging in DIV10 neurons (1 frame every 5 seconds for 10 minutes). We discovered, that at DIV10, lack of Panx1 KO stabilized spiny protrusions. Notably, re-expression of Panx1 in Panx1 knockout neurons resulted in a significant increase in spiny protrusion motility and turnover. In summary, these new data revealed that Panx1 regulates the development of dendritic spines by controlling protrusion dynamics. Significance statement Cells in the brain form intricate and specialized networks - neuronal networks - in charge of processing sensations, executing movement commands, and storing memories. To do this, brain cells extend microscopic protrusions - spiny protrusions - which are highly dynamic and survey the local environment to contact other cells. Those contact sites are known as synapses and undergo further stabilization and maturation establishing the function and efficiency of neuronal networks. Our work shows that removal of Panx1 increases the stability and decreases the turnover of spiny protrusion on young neurons.


Introduction
Pannexin 1 (Panx1) is a four transmembrane domain protein that forms channels permeable to ion and metabolites with various activation mechanisms and diverse (patho)physiological implications (for review Boyce et al., 2018;Chiu et al., 2018). Panx1 is broadly and highly expressed in the brain during postnatal early development (Ray et al., 2005;Vogt et al., 2005) and localized and enriched in synaptic compartments (Sanchez-Arias et al., 2019;Zoidl et al., 2007).
Recent reports have implicated Panx1 in neurite outgrowth, hippocampal synaptic plasticity, and the development of neuronal networks and dendritic spines in cortical neurons (Ardiles et al., 2014;Prochnow et al., 2012;Sanchez-Arias et al., 2019;Wicki-Stordeur & Swayne, 2013). While the behavioural features resulting from a loss of Panx1 have not been thoroughly characterized, a handful of studies have detected important phenotypes like anxiety, increased wakefulness, and spatial learning deficits (Ardiles et al., 2014;Gajardo et al., 2018;Kovalzon et al., 2017;Prochnow et al., 2012). Notably, dendritic spine development has been linked to each of these behaviours. For example, dendritic spine density is increased in various neurodevelopmental disorders in which clinical manifestations include anxiety, intellectual disability, and stereotypical movements (Phillips & Pozzo-Miller, 2015).
Dendritic spine-based synapses result from spiny protrusions (including dendritic filopodia) actively extending to contact presynaptic boutons during developmental excitatory synaptogenesis; upon contact, spiny protrusions stabilize and evolve into mature dendritic spines along active presynaptic boutons (Fiala et al., 1998;Ziv & Smith, 1996). These steps are critical in establishing network ensembles and Hebbian plasticity (Hoshiba et al., 2017).
In light of this evidence, we investigated the role of Panx1 in spiny protrusion dynamics in cultured primary cortical neurons at 10 days-in-vitro (DIV10).
We first established an approach to study spiny protrusions using a fluorescent membrane tag (mCherry-CD9-10), allowing us to visualize these characteristically long and thin structures. Then, we transiently transfected wildtype (WT) and Panx1 knock-out (KO) neuronal cultures with EGFP or Panx1EGFP (as well as mCherry-CD9-10) and analyzed spiny protrusions in fixed and living neurons at DIV10. We confirmed that lack of Panx1 leads to higher spiny protrusion density while over-expression and rescue of Panx1 leads to decreased density. Using live cell imaging we observed increased stability and decreased turnover of spiny protrusions in Panx1 KO neurons, while re-expression of Panx1 resulted in a significant increase in spiny protrusion motility and turnover. In summary, these new data reveal an inverse relationship between Panx1 expression and dendritic spine stability.  (Bardy et al., 2015); to limit proliferation of glial cells, ara-C was added to the medium at DIV4. and exported to FIJI for analysis (Schindelin et al., 2012). Individual spiny protrusions were traced along the longest neurite (primary neurite) and their density was calculated by dividing the total number of spiny protrusions by the segment length and multiplying by 10 (spiny protrusions per 10 µm). Representative images were processed uniformly with a Gaussian blur of 0.5 pixels, and uniform adjustments to levels and contrast were made using Photoshop CS6

Materials and Methods
Extended suite (Adobe Systems).

Imaging and analysis of spiny protrusions in live cortical neurons
Cortical neurons plated on chambered coverglasses in BrainPhys™ at 37°C and 5% CO2 and primary and secondary dendrite segments of 67-76 µm were imaged (1024×256, pixel size: 0.06 µm) every 5 seconds for 10 minutes and 0.7 μm z-step using a Leica TSC SP8 microscope in resonant mode (8,000 Hz) with a 63× water immersion objective (1.20 NA).
Images were exported to FIJI for analysis. First, the four-dimensionality (x,y,z,t) was reduced by creating maximum z projections before additional image processing and x-y drift was corrected with Multi-StackReg v1.45 (developed by Brad Busse http://bradbusse.net/MultiStackReg1.45_.jar) when required. Then, images were subjected to a low-pass filter using a Gaussian blur (kernel size 2) and thresholded using the triangle method (Zack et al., 1977). From these binary images, outlines for each time frame were created and temporal colour-coded ( Figure 3A,B). Spiny protrusions were manually counted, and four basic characteristics were recorded: formation, elimination, lability, and motility. We

Experimental design and statistical analysis
For all experiments 3 independent cultures were used. All images were blindly acquired and analyzed.

Measuring spiny protrusion dynamics in living neurons using a membrane marker
To investigate the mechanisms contributing to differences in spiny protrusion densities between groups, we acquired 10-minute time-lapses (one frame every 5 seconds) of primary and secondary dendrites from cortical neurons at DIV10. These cultures were transfected with mCherry-CD9-10 and either EGFP or Panx1EGFP at DIV6. At DIV10, dendrites harbour highly dynamic, thin, and long spiny protrusion that are the precursors for dendritic spines (Fiala et al., 1998;Ziv & Smith, 1996). We reduced the dimensionality of the time-lapses by creating maximum z-projections, and then images were passed through a low-pass filter and thresholded to create outlines ( Figure 3A). The dendritic silhouettes ( Figure 3B) were then temporally colour-coded to facilitate the detection of formation, elimination, liability, retraction, and growth of spiny protrusions ( Figure 3C).

Basic characteristics of spiny protrusion dynamics in WT and Panx1 KO neurons at DIV10
Using the above approach, we observed that trans-   Effect of Panx1 expression on spiny protrusion formation, elimination, lability, and motility in WT and Panx1 KO using Cumming estimation plots. We used the basic characteristic measurements devised in Figure     Consistent with this idea, recent work has identified brain enriched and autism associated single nucleotide polymorphisms (SNPs) resulting in changes in Panx1 expression levels; although the direction of this change (i.e. decrease or increase Panx1 expression) was not identified (Davis et al., 2012). Further supporting a role for Panx1 in neuronal development, intellectual disability was observed in an individual with a germline single nucleotide polymorphism in PANX1 (Shao et al., 2016).

Bi. Spiny protrusion formation was significantly higher in
In addition to playing a direct role in neurodevelopment, Panx1 is also indirectly involved through its interaction with Crmp2 and purinergic receptor signalling (Boyce et al., 2015;reviewed in Swayne & Boyce, 2017). Crmp2 auto-antibodies have been implicated in ASD (Braunschweig et al., 2013), while suramin treatment corrected synaptic and behavioural phenotypes in the Fragile X mouse model (J. C. Naviaux et al., 2015;R. K. Naviaux et al., 2013R. K. Naviaux et al., , 2017. In summary, this work significantly advances our understanding of the role on Panx1 in dendritic spine development and underscores the importance of additional molecular mechanistic studies investigating intrinsic (e.g. Crmp2) and extrinsic (e.g. glia) pathways.