Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

The muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein-coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking. We overexpressed M1R in primary cultures of adult rat sensory neurons and cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. In adult primary neurons, overexpression of M1R caused disruption of the tubulin, but not actin, cytoskeleton and significantly reduced neurite outgrowth. Over-expression of a M1R-DREADD mutant comparatively increased neurite outgrowth suggesting that acetylcholine released from cultured neurons interacts with M1R to suppress neurite outgrowth. M1R-dependent constraint on neurite outgrowth was removed by selective (pirenzepine) or specific (muscarinic toxin 7) M1R antagonists. M1R-dependent disruption of the cytoskeleton also diminished mitochondrial abundance and trafficking in distal neurites, a disorder that was also rescued by pirenzepine or muscarinic toxin 7. M1R activation modulated cytoskeletal dynamics through activation of the G protein (Gα13) that inhibited tubulin polymerization and thus reduced neurite outgrowth. Our study provides a novel mechanism of M1R control of Gα13 protein-dependent modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. This novel pathway could be harnessed to treat dying-back neuropathies since anti-muscarinic drugs are currently utilized for other clinical conditions.

The muscarinic acetylcholine type 1 receptor (M 1 R) is widely expressed in the central nervous system (CNS) (Levey, 1993;Wess et al., 2003;Jiang et al., 2014) and peripheral nervous system (PNS) (Bernardini et al., 1999;Tata et al., 2000b). Membranes isolated from hippocampus and cortex of M 1 R knockout (KO) mice showed a significant decrease in GTPγ-S loading to the Gα-q/11 G protein upon agonist stimulation (Felder et al., 2001). In cortical neuron cultures obtained from M 1 R KO mice, carbachol-stimulated phosphoinositide hydrolysis was reduced by 60% compared with wild type (Bymaster et al., 2003). In addition, phosphorylation of extracellular signal-regulated kinase (ERK) was eliminated in pyramidal neurons of hippocampal slices or cortical cultures derived from M 1 R KO mice (Berkeley et al., 2001;Hamilton and Nathanson, 2001).
We have recently reported that selective or specific antagonists of M 1 R elevated neurite outgrowth and augmented mitochondrial function in adult sensory neurons (Calcutt et al., 2017). These drugs also afforded protection against several different forms of peripheral neuropathy. However, the mechanism of M 1 R antagonist-driven neurite outgrowth and neuroprotection has not been studied in detail. Mitochondrial oxidative phosphorylation is the main mechanism providing ATP to power neuronal activities such as production of presynaptic action potentials, neurotransmitter release, postsynaptic currents and postsynaptic action potentials (Hall et al., 2012). Mitochondria are known to concentrate in regions of active signaling and high metabolic demand (Chen and Chan, 2006;Mironov, 2007;Verburg and Hollenbeck, 2008). This substantial energy demand at the nerve ending or synapse implies that neurons must have a mechanism to maintain microtubules to augment mitochondrial trafficking upon demand (Sheng and Cai, 2012;Schwarz, 2013).
In the present study we manipulated M 1 R expression/function in adult DRG sensory neurons and related cell lines and studied the cellular phenotypes and molecular consequences. Specifically, we tested the hypothesis that the M 1 R regulates the tubulin cytoskeleton, G-protein recruitment (Gα13 subtype) and mitochondrial trafficking. We identified that excessive cholinergic signaling triggered tubulin destabilization through over-activation of Gα13 proteins. Further, we studied the ability of specific (muscarinic toxin 7: MT7) or selective (pirenzepine) M 1 R antagonists to ameliorate the endogenous and M 1 R overexpression-induced neuronal phenotypes that primarily result in a constraint on neurite outgrowth.

MATERIALS AND METHODS
All animal procedures followed guidelines of University of Manitoba Animal Care Committee using Canadian Council of Animal Care rules or of the Institutional Animal Care and Use Committee at UCSD.
HEK293 and HTLA cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated FBS. The β-arrestin null (ARRB1 and ARRB2) and Gα12/13 (GNA12 and GNA13) null HEK293 cells were obtained from the laboratory of Dr. Asuka Inoue, Tohoku University, Japan. HTLA cells were provided by Prof Bryan Roth, University of North Carolina, United States.

Cloning, Transfection and siRNA Based Gene Silencing
Total mRNA was extracted from adult rat DRGs using Trizol reagent and used for amplifying the full length M 1 R cDNA using the following primer sets: F: 5 -ATGAACACCT CAGTGCCCCCTGC-3 and R: 5 -TTAGCATTGGCGGGAG GGGGTG-3 . The cDNA was cloned in the pEGFP-C1 vector (Clontech, now Takara Bio United States, Inc., Mountain View, CA, United States) in the XhoI and SacII restriction sites. In addition, the cDNA was also cloned in the pHTN Halo-Tag CMV-neo Vector (Promega, Madison, WI, United States) at the SacII and Not1 restriction sites. The plasmids were transiently transfected in freshly dissociated sensory neurons using the rat neuron nucleofection kit (VPG-1003, Amaxa, Lonza Inc., Allendale, NJ, United States) and Amaxa nucleofector-II apparatus (program 0-003) and cultured in poly L-Ornithine (P8638, Sigma) and laminin coated µ-Plate-24 well (Ibidi United States, Inc., Madison, WI, United States). The human M1-DREADD construct was obtained from Dr. Arthur Christopoulos, Monash University, Australia and sub-cloned in pEGFP-C1 vector (Abdul-Ridha et al., 2013). The rat Gα13(GNA13) was knocked down using a cocktail of three siRNAs targeted to exon 2 (AGTATCTTCCTGCTATAAGAGCC) and exon 4 (CTACAT TCCGTCACAGCAAGATA and CATCAAAGACTATTTCCTA GAAT), respectively. The siRNAs were transfected in to primary sensory neurons using Amaxa transfection reagent.

Quantification of Neurite Outgrowth
The transgene transfected neurons were cultured for 48 h and then cells were fixed in 4% paraformaldehyde for 10 min and immunostained using monoclonal anti-β-tubulin III antibody. The neurons were also stained with Hoechst for nuclear staining. The neurons were imaged in an unbiased manner using a Cellomics Arrayscan-VTI high content screening (HCS) Reader (Thermo Fisher Scientific, Waltham, MA United States) and total neurite outgrowth per neuron was measured by Neuronal Profiling V4.1 software. The automated HCS reader provided a bias-free objective analysis of neurite outgrowth.

Confocal Microscopic Image Acquisition and Analysis to Determine Mitochondrial Volume and Trafficking
Mitochondrial trafficking in GFP or GFP-M 1 R overexpressing neurons was monitored using LSM510 confocal live cell imaging and involved co-transfection of sensory neurons with GFP/GFP-M 1 R and DsRed2Mito7 plasmids (Addgene plasmid #55838, a gift from Dr. Michael W. Davidson, Florida State University), respectively. The DsRed2Mito7 consists of a mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase which is placed before the Red fluorescence protein and the resultant fusion protein selectively accumulates inside the mitochondria (Van Kuilenburg et al., 1988). The transfected cells were live imaged at 10 s interval for 80 time frames (∼13 min). The time lapse images were used to generate kymographs using the ImageJ make kymograph plugin (Schneider et al., 2012).
In each kymograph, the x-axis represents the position along the length of the axon and the y-axis represents time. Vertical lines indicate stationary mitochondria with no displacement during the time elapsed and diagonal lines represent moving mitochondria and their direction. Their velocity is reflected in the slope of the line. In addition we used Fiji (Schindelin et al., 2012) based MTrackJ to determine mitochondrial velocity (µm/sec) in the neurites (Meijering et al., 2012). The volume of mitochondria in the neurites was calculated by using Image J analyze particles plugin and expressed in µm per neurite length (Schindelin et al., 2012).

Western Blotting and Immune-Detection
Relative quantification of proteins was done by SDS-PAGE separation of total proteins followed by transfer to nitrocellulose membrane and immunoblotting based detection using HRPconjugated secondary antibodies. The immunoblots were imaged in Bio-Rad Chemidoc system (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada).

Polymerized Tubulin Quantification
The polymerized tubulin in M 1 R overexpressed cells (40-50% transfection efficiency) was quantified by methods described previously (Butcher et al., 2016). Briefly, the soluble fraction of tubulin was first removed by lysing the cells in a microtubule stabilizing buffer (MSB) containing 50% glycerol, 5 mM MgCl 2 , 0.1 mM EGTA, 0.3 mM guanosine triphosphate (grade II-S, Sigma Chemical Co.), and 10 mM sodium phosphate, pH 6.8  (Beertsen et al., 1982). Cells were harvested with a rubber scraper in MSB, homogenized, and centrifuged at 26,000 × g in a Sorvall RC2-B centrifuge (17,000 rpm in rotor SS-34 in 1.0 ml tubes; Dupont Instruments-Sorvall Biomedical Div., Dupont Co., Newtown, CT, United States) at 20 • C for 30 min. Supernatants containing the soluble tubulin fraction were removed and the pellet containing polymerized tubulin and other cytoskeletal protein was assessed by immunoblotting using anti-actin and anti-β tubulin III antibodies.

Isoelectric Focusing
Fifty microgram of total cellular protein was precipitated by acetone and dissolved in rehydration buffer containing 8 M Urea, 2% CHAPS, 50 mM dithiothreitol (DTT) and 0.2% Bio-Lyte ampholytes pH3-10. The dissolved proteins were incubated in Zoom IPG-strip 3-10 non-linear (NL) (Thermo Fisher) for 1 h and then focused at 175 volt (V) for 15 min, 175-2000 V ramp for 45 min and 2000 V for 30 min. After the run, the strips were alkylated and resolved on 2D SDS-PAGE and immunoblotted using antibodies previously described.

Statistical Analysis
Statistical analysis was performed using Prism version 7.00 (GraphPad Software). The mean of two or more groups were compared using one-way ANOVA followed by multiple comparison tests (Siegel, 1956;Dunn, 1964). The mean of multiple experimental groups were compared with the control group by Dunnett's post hoc multiple comparison test, whereas, the mean difference between two experimental groups were compared by Sidak's post hoc multiple comparison test (Dunn, 1964). Comparisons between two groups were performed using Student's t-test (unpaired). Differences were considered significant at P < 0.05. Neurite outgrowth, neurite width and mitochondrial quantification data were plotted as box and whisker plot where the end of the box represents the upper and lower quartiles and the median is marked by a horizontal line inside the box. The whiskers represent the highest and lowest values excluding the outliers. In some figures the individual data points of outliers are also indicated.

MT7 and Pirenzepine Significantly Augmented Neurite Outgrowth in Cultured Primary Sensory Neurons
Pirenzepine is a selective M 1 R antagonist whereas MT7 is the only specific antagonist of this receptor (Birdsall et al., 1983;Max et al., 1993a). Our previous study demonstrated that both antagonists enhanced neurite outgrowth from adult sensory neurons (Calcutt et al., 2017). In order to confirm that MT7 and pirenzepine had growth promoting effects under the current conditions, we measured total neurite outgrowth from primary sensory neurons derived from adult rats and cultured in defined media containing a cocktail comprising of low or high concentrations of GDNF, NGF, and NT3 growth factors (LGF and HGF, respectively) and either 100 nM MT7 or 1 µM pirenzepine (Figures 1A-D). The growth factor concentrations in the LGF cocktail reflects concentrations of growth factors that are sub-saturating and induce small but significant increases in neurite outgrowth (Calcutt et al., 2017). The HGF cocktail contained 10-fold higher concentrations to allow us to determine whether the neuritogenic effects of MT7 and pirenzepine were also effective when growth factors were present in excess and, also to see if increased tyrosine kinase signaling masked the effect of antagonist-M 1 R signaling mediated growth. In the absence of muscarinic antagonists, the HGF promoted significantly (p < 0.0001) more neurite outgrowth than LGF, as assessed using unbiased automated high content imaging combined with data analysis (Figures 1A,B). Both MT7 and pirenzepine significantly increased neurite outgrowth in LGF and HGF conditions within 48 h of treatment ( Figures 1A,B).
In addition, binning of the entire data set of 1249 (LGF) and 1517 (HGF) neurons for their total neurite length revealed that the neuritogenic effect of pirenzepine and MT7 evenly affected neurite outgrowth of the majority of the population of neurons (Figures 1C,D). Further, pirenzepine treatment elicited significantly higher neurite outgrowth under the LGF conditions when compared to MT7, whereas under the HGF condition, the primacy was reversed (Figures 1A,B). The exact reason for the difference between the drugs in terms of synergistic effect on growth at HGF condition is not known. The difference in the chemical nature of these drugs may be responsible. MT7 is a cell impermeable 7.4 kDa protein (Krajewski et al., 2001) which binds allosterically to M 1 R (Max et al., 1993b;Karlsson et al., 2000) whereas pirenzepine is a cell-permeable orthosteric antagonist molecule (Caulfield and Birdsall, 1998). We used LGF conditions in subsequent experiments.

M 1 R Overexpression Induced Significant Reduction in Neurite Outgrowth
The muscarinic receptor subtypes (M1-M5) show considerable heterogeneity of expression in sensory neurons (Bernardini et al., 1999;Chiu et al., 2014). In order to understand the biological function of M 1 R in sensory neurons, we overexpressed GFP-tagged M 1 R in sensory neurons and measured the impact on neurite outgrowth. M 1 R overexpression significantly reduced neurite outgrowth when compared to GFP-expressing neurons (Figures 1H,I). The GFP-M 1 R transgene-induced protein production was verified in the transfected DRG neurons by immunoblotting of the expressed recombinant proteins (GFP-M1-5R) using both anti-M 1 R and anti-GFP antibodies ( Figure 1E). The biological functionality of the GFP-M 1 R recombinant protein was verified by treating GFP-M 1 R overexpressing DRG neurons with the broad spectrum muscarinic agonist carbachol (10 µM) followed by live confocal imaging to monitor internalization of recombinant protein. Treatment with carbachol increased presence of the recombinant protein as internalized spots that were positive for the early endosomal marker Rab5 (Zerial and McBride, 2001) ( Figures 1F,G). This indicates that GFP-tagged recombinant M 1 R elicited agonist induced internalization response similar to that of naïve proteins. In addition, to assess functionality of the recombinant M 1 R in overexpressed cells, we performed a β-arrestin recruitment assay [TANGO assay: (Kroeze et al., 2015), Figure 2]. In the TANGO assay, upon activation, β-arrestin is recruited to the C-terminus of the M 1 R-TEV-tTA fusion protein at the TEV protease site and cleaves to release the tTA transcription factor, which after transport to the nucleus activates transcription of luciferase reporter gene (Kroeze et al., 2015) (Figure 2A). We verified expression of the M 1 R-TEV-tTA and β-arrestin transgenes by immunoblotting ( Figure 2B). We found significant recruitment of β-arrestin due to basal M 1 R activation in presence of serum in the culture media in the HTLA cells ( Figure 2C). This basal activation of M 1 R occurred in the absence of exogenously supplied agonist. However, fetal bovine serum (FBS) used in the culture media is reported to contain acetylcholine (ACh) (Lau et al., 2013) and this may have constitutively acted upon the M 1 R. Treatment with the muscarinic agonist, carbachol, significantly increased activation of the M 1 R by up to ∼7.4-fold ( Figure 2C).

Endogenous ACh Binds M 1 R to Constrain Neurite Outgrowth
Adult sensory neurons secrete ACh into the extracellular media when grown in culture (Calcutt et al., 2017). The Tango Assay revealed that ACh present in the serum supplied to the culture media may also act upon M 1 R and constitutively activate the receptor. We therefore hypothesized that overexpression of M 1 R in sensory neurons may lead to increased constitutive activation of the receptor by recruitment of trimeric G-proteins, including the Gα12/13 subtype that promotes tubulin destabilization (Roychowdhury et al., 1999). In order to test this hypothesis we transfected DRG neurons with an M 1 R-DREADD (designer receptors exclusively activated by designer drugs) mutant to eliminate the putative basal activity in M 1 R-DREADD overexpressing neurons (Figures 2D,E). The M 1 R-DREADD mutant contained two mutations in the conserved orthosteric site residues (Y106C and A196G in the M 1 R) that minimize responsiveness to ACh (Abdul-Ridha et al., 2013). Overexpression of GFP-M 1 R and M 1 R-DREADD caused low levels of neurite outgrowth (Figures 2D-F). However, neurite outgrowth in M 1 R-DREADD neurons was significantly higher than GFP-M 1 R alone over 72 h of culture (Figures 2D-F). This indicates that endogenous ACh may interact with M 1 R to limit neurite outgrowth.

Impact of M 1 R Overexpression on Mitochondrial Abundance
In an attempt to understand the mechanism of the growth inhibitory effect of M 1 R overexpression, we examined the abundance of mitochondria in the neurites by co-expressing DsRed2Mito7 plasmid ( Figure 3A). The DsRed2Mito7 plasmid expresses DsRed protein tagged with mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase and therefore localizes specifically to mitochondria. We measured the volume of mitochondria present per unit length (µm) of neurites in the M 1 R and GFP overexpressing DRG neurons ( Figure 3B). M 1 R overexpression significantly reduced the abundance of mitochondria in the neurites after 48 h of culture (Figures 3C,D). The mitochondria in both M 1 R and GFP over-expressing neurons accumulated mitotracker CMXRos dye, indicating that functionality of the mitochondria was not impaired in M 1 R overexpressed neurons despite reduced abundance (data not shown).

Reduced Mitochondrial Abundance Was Associated With Impaired Cytoskeletal Structure
To determine whether reduced mitochondrial abundance was related to a defect in the actin or tubulin cytoskeletons, we immunostained DRG neurons that overexpressed GFP/GFP-M 1 R with phalloidin and anti-β-tubulin III antibodies. In the presence of M 1 R over-expression, immunofluorescent imaging of phalloidin revealed the presence of abundant and continuous actin filaments in the neurites, although the neurite tips were notably thinner compared to those of GFP expressing neurites ( Figure 4A). In contrast, the β-tubulin III associated cytoskeleton appeared less abundant and discontinuous ( Figure 4B). Fragmentation of the tubulin cytoskeleton was confirmed using a polymerized tubulin quantification assay in which there was significantly less polymerized tubulin in the DRG neurons overexpressing M 1 R compared to those that overexpressed only GFP (Figures 4C,D).

Discontinuous Tubulin Cytoskeleton Was Associated With Reduced Mitochondrial Trafficking in Sensory Neurons
Overexpressing M 1 R In order to determine whether the discontinuous β-tubulin cytoskeleton was associated with altered mitochondrial trafficking in growing neurites, we measured velocity of mitochondrial movement in the neurites of DRG neurons that co-expressed GFP/GFP-M 1 R and DsRed2Mito7. Time-lapse images of the neurites at 10 s intervals were used to generate kymographs of mitochondrial trafficking ( Figure 5A). The mean velocity of mitochondria was calculated as 1.9 µm/sec in GFP expressing neurons whereas in GFP-M 1 R expressing neurons it was significantly lower at 0.62 µm/sec (Figures 5B,C). In addition, immunostaining of neurites with a similar appearance to those used in the mitochondrial velocity measurement revealed a continuous β-tubulin-III cytoskeleton in GFP over-expressed neurons and a discontinuous β-tubulin-III cytoskeleton in M 1 R over-expressed neurons (Figures 5D,E).

Muscarinic Antagonists MT7 and Pirenzepine Rescued the Cytoskeletal Defect, Aberrant Mitochondrial Distribution and Trafficking in Neurites
We investigated whether pirenzepine or MT7 could overcome M 1 R overexpression-induced cytoskeletal defects. M 1 R overexpressing neurons were maintained for 48 h and then treated for 24 h with 100 nM MT7 or 1 µM pirenzepine and total neurite outgrowth and mitochondrial velocity ( Figure 5) and abundance (Figure 6) quantified. MT7 and pirenzepine treatment significantly rescued the deficits in the mitochondrial velocity in M 1 R overexpressed neurons, with mean mitochondrial velocities of 0.78/1.2 µm/s in MT7/pirenzepine treated neurons being significantly higher than untreated neurons (Figures 5B,C). Both MT7 and pirenzepine also caused considerable re-localization of M 1 R from the perikarya to the neurites, as revealed by time lapse confocal live cell images over a period of 72 h. This may indicate increased vesicular transport of internalized M 1 R ( Figure 6A). Within the same neuron (shown in Figure 6A), following 24 h of drug treatment and upon fixation and immunostaining for β-tubulin III, there was continuity in the β-tubulin III associated cytoskeleton (right panel). MT7 or pirenzepine significantly increased total neurite outgrowth, reversed the reduced neurite caliber and increased mitochondrial abundance in DRG neurons that overexpressed GFP-M 1 R (Figures 6B-E).

Knockdown of Gα13 in Sensory Neurons Reversed the M 1 R Overexpression-Induced Tubulin Cytoskeleton Defect
The M 1 R-DREADD mutant study raised the possibility that, in normal DRG neurons, basal M 1 R activity resulting from binding of endogenous ACh release may destabilize the tubulin cytoskeleton through increased active G proteins. Gα12/13 proteins are known for their effect on cytoskeleton remodeling and the M 1 R receptor activates Gα12/13 type G proteins leading to mobilization of the small GTP-binding protein Rho through activation of Rho-GEF (RhoGTPase nucleotide exchange factor) (Haga, 2013). We, therefore, measured the relative expression of Gα12 and Gα13 proteins in sensory neurons ( Figure 7A). Immunoblots revealed that DRG neurons express significantly more Gα13 compared to Gα12 (Figures 7A,B). The relative levels of Gα12 and Gα13 expression were comparable to those of the human carcinoma cell line HEK293 (Supplementary Figure S1A). We used siRNA to knockdown Gα13 in DRG neurons and used a CRISPR/Cas9 based Gα12/13 null HEK293 cell line to study the effect of cholinergic signaling through Gα13 on the tubulin cytoskeleton ( Figure 7C and Supplementary Figure S1A). The HEK293 cell line has been reported to exhibit a neuronal lineage phenotype and express neuronal proteins (Stepanenko and Dmitrenko, 2015). Therefore, we considered it suitable for this study. The siRNA based knockdown of Gα13 in DRG neurons had no significant effect on neurite outgrowth (Figures 7D,E). Interestingly, knockdown of Gα13 in M 1 R overexpressed sensory neurons significantly reversed the suppressed neurite outgrowth (Figures 7F,G). In addition, treatment of the M 1 R overexpressed and Gα13 knockdown neurons with 100 nM MT7 or 1 µM pirenzepine exhibited significantly increased neurite outgrowth (Figures 7F,G).

CRISPR/Cas9 Based Gα12/13 Null HEK293 Cells Showed Abundant Tubulin Cytoskeleton but Diminished Actin Stress Fibers
RNA sequencing data in the human protein atlas (HPA) indicates that HEK293 cells express very high levels of α-tubulin compared to β-tubulin III (Uhlen et al., 2005). The transcripts per million bases (TPM) value for α-tubulin (TUBA1B isoform) and β-tubulin III has been recorded as 1923.6 and 13.7, respectively 1 . β-tubulin III was undetectable by immunoblotting in HEK293 cells (data not shown). We, therefore, studied α-tubulin dynamics in CRSIPR/Cas9 based Gα12/13 knockout HEK293 cells. We performed immunofluorescent labeling using phalloidin and anti-α-tubulin specific antibodies to visualize the actin and tubulin based cytoskeletal structures in Gα12/13 knockout HEK293 cells (Supplementary Figure S1E). Phalloidin staining revealed that actin stress fibers were diminished in Gα12/13 null cells, with appearance of abundant distinct punctate actin rich focal adhesion points (Supplementary Figure S1E). Further, the tubulin cytoskeleton appeared more robust and organized in Gα12/13 null cells (Supplementary Figures S1E, S2). The BN-PAGE based microtubule fractionation assay and polymerized microtubule quantitative assay showed Gα12/13 null cells exhibited significantly more polymerized tubulin than wild type HEK293 cells (Supplementary Figures S1B-D). Overexpression of M 1 R in Gα12/13 null cells and treatment with muscarinic agonist carbachol did not alter the tubulin networks as compared to wild type cells (Supplementary Figures S2, S3).  (Yamada et al., 2011). We examined the recruitment of trimeric G proteins to M 1 R by Halo-pull down assay and BN-PAGE analysis (Figures 8, 9). The Halo-pull down assay permitted a focus on the over-expressed M 1 R with no contamination from endogenous muscarinic receptors of mixed sub-type. The SH-SY5Y cells that overexpressed Halo-M 1 R were treated with drugs and Halo-tagged M 1 R was pulled down using Halo-linked resin. Subsequently, the pull down product was resolved in SDS-PAGE and immunoblotted with anti-M 1 R, anti-Gα12/13 and anti-Gγ/2/3/4/7 antibodies (Figures 8B-D). The Halo-M 1 R pull down fraction from the drug treated cells showed significantly elevated levels of Gγ/2/3/4/7 and Gα12/13 proteins when compared with untreated cells (Figures 8E,F). The 2D BN-PAGE/SDS-PAGE analysis revealed existence of 2 protein complexes at ∼900 and ∼1200 kDa equivalent molecular weights in Halo-M 1 R over-expressing SH-SY5Y cells (Figures 9A,B). Each complex was associated with a native ∼100 kDa Halo-tagged M 1 R protein and >180 kDa fractions, the latter may be derived from different PTMs of the Halo-M 1 R. Treatment with 100 nM MT7 caused a major shift of the ∼900 kDa protein complex to the ∼1200 kDa protein complex within 1 h of treatment, suggestive of recruitment of putative interacting proteins (Figure 9, blue and red dotted areas). In contrast, untreated cells did not show any shift in the ∼900 kDa protein complex to the ∼1200 kDa protein complex indicating less or no recruitment of interacting proteins ( Figure 9A). Further, when the same blots were immunoblotted with anti-Gα12/13 antibodies, the Gα12/13 proteins appeared as spots on a vertical line corresponding to the ∼1200 kDa protein complex in the drug treated cells which suggests possible co-migration and association with M 1 R (Figures 9A,B, bottom panel).

DISCUSSION
Our recent work has shown that sensory neurons derived from M 1 R null mice exhibit enhanced neurite outgrowth (Calcutt et al., 2017). We now demonstrate that over-expression of M 1 R inhibited neurite outgrowth, caused disruption of the tubulin cytoskeleton and blockade of mitochondrial trafficking in adult sensory neurons, all of which were rescued by exposure to selective or specific M 1 R antagonists. We then used overexpression of GFP-M 1 R to identify the molecular pathway components associated with specific M 1 R-mediated cellular phenotypes. Based on our data, we propose that ACh mediated signaling via M 1 R constrains neurite outgrowth via activation of Gα13 proteins, which in turn limits tubulin polymerization and mitochondrial trafficking within axons.
Overexpression of M 1 R in sensory neurons has biological consequences that likely arise from the presence of neuronderived ACh in the culture environment. Cultured sensory neurons secrete endogenous ACh into the extracellular media to generate a local concentration in the range of approximately 16 µM (Calcutt et al., 2017). This far exceeds the ACh K d of 25-35 nM measured in several regions of rat brain tissue (Kellar showing the relative amount of G proteins (Gα and Gγ, respectively) associated with M 1 R following drug treatment. The data represent mean ± SEM of three independent experiments. p-values ( * * * * <0.0001) were calculated by one-way ANOVA with Dunnett's post hoc multiple comparison test. et al., 1985) or the K d of 0.2-0.4 nM measured using rat brain neurons (Pavia et al., 1991;Bakker et al., 2015). In the presence of abundant extracellular ACh, overexpressed. M 1 R will trigger activation of Gα13 proteins that leads to the dissociation of tubulin microtubules, as seen in Figure 4. The chemogenetically modified M 1 R-DREADD mutant significantly reduced M 1 R induced growth retardation (Figure 2). Thus, ACh-driven basal activity of M 1 R is responsible for neurite outgrowth suppression under conditions of M 1 R over-expression. In addition, we propose in normal un-transfected neurons that basal endogenous M 1 R signaling tonically suppresses neurite outgrowth by restricting mitochondrial (and potentially vesicular) transport, thereby explaining the ability of antimuscarinic drugs to prevent/reverse this constraint.
Knockdown of Gα13 in M 1 R-overexpressed neurons significantly reversed the M 1 R-induced growth inhibitory effect and protected from tubulin destabilization in growth cones (Figure 7). Gα13 is more abundant than Gα12 in sensory neurons and M 1 R was linked to activation of Gα12/13 type G proteins that leads to activation of small GTP binding protein Rho through mobilization of RhoGEF (Luo et al., 2001;Siehler, 2009;Haga, 2013). Activation of Gα12/13 leads to stimulation of the Rho/Rho Kinase pathway via a subgroup of Rho guanine nucleotide exchange factors (Fukuhara et al., 2001). Involvement of Rho and ROCK (Rho-associated coiled coil forming protein kinase) in agonist-induced neurite retraction and cell rounding has been reported in N1E-115 neuroblastoma cells (Hirose et al., 1998). Dominant-negative p160-ROCK completely abolished this neurite retraction suggesting a clear link between RhoA-ROCK signaling and cytoskeleton disassembly (Hirose et al., 1998). Activation of neuronal cannabinoid receptors linked to Gα12/13 proteins triggered rapid and reversible contraction of actinomyosin cytoskeleton through a Rho-GTPase and ROCK (Roland et al., 2014). Further, the CRISPR/Cas9 based knockout of Gα12/13 proteins in the HEK293 cell line augmented the abundance of polymerized tubulin (Supplementary Figure S1). HEK293 cells have been utilized as a model system for neuronal synapse formation (Biederer and Scheiffele, 2007) as they express neuronal proteins and have neuronal cell-lineage (Shaw et al., 2002;Stepanenko and Dmitrenko, 2015). Using a NanoBIT split luciferase based RhoA biosensor that detects Gq-induced RhoA activation showed that the RhoA signal is completely lost in the Gα12/13 KO cell (Mercier et al., manuscript in revision, personal communication).
Activated G proteins regulate tubulin polymerization (Schappi et al., 2014) and tubulin binds directly to Gα or Gβγ subunits (Wang et al., 1990;Roychowdhury et al., 1999;Sarma et al., 2003). Activated GTP bound Gα promotes microtubule instability by increasing the intrinsic hydrolysis of GTPtubulin to the less polymer stable GDP-tubulin (Roychowdhury et al., 1999(Roychowdhury et al., , 2006. We have modelled this in Figure 10. Overexpression of Gα q in a rat pituitary cell line showed a 50% decrease in the ratio of soluble to polymerized tubulin (Ravindra et al., 1996). There is considerable cell type and isoform specificity in Gα mediated tubulin cytoskeleton dynamics (Sarma et al., 2015). We found that Gα12/13 and Gγ(2,3,4,7) were sequestered upon M 1 R antagonist binding (Figures 8, 9) and it is plausible that these factors also regulate tubulin polymerization in sensory neurons. In addition, some guanine nucleotide exchange factors (GEFs) for Rho GTPases, namely p115 RhoGEF (Kozasa et al., 1998), PDZ-RhoGEFs (Fukuhara et al., 1999), and LARG (Suzuki et al., 2003) can act as direct couplers of Gα12/13 proteins to small GTPases such as RhoA, Rac1, and CDC42, all of which are known to influence microtubule dynamics (Hall and Lalli, 2010). The Gα12/13-RhoGEF-RhoA pathway of GPCR has been implicated in many diseases (Siehler, 2009). Interestingly, while Gα promotes tubulin disassembly by increasing the tubulin GTPase activity, Gβγ subunits preferentially associate with GDP-bound tubulin to promote polymerization and stability of the microtubule (Roychowdhury and Rasenick, 1997;Popova and Rasenick, 2003;Roychowdhury et al., 2006). Our data from non-crosslinked halo-pull down (Figure 8) and BN-PAGE analyses (Figure 9) show augmented association and occupancy of trimeric G-proteins (Gα12/13) to M 1 R during MT7 or PZ treatment, which indicates elevated sequestration of these proteins on M 1 R. We propose that muscarinic antagonist-induced sequestration of trimeric G-proteins restricts their dissociation from the overexpressed M 1 R associated protein complex and thereby limits their detrimental effect on tubulin polymerization (see Figure 10). Further, we posit that the same M 1 R suppression pathway is occurring in normal un-transfected neurons to limit cytoskeleton formation in axons and is counteracted by these drugs. However, this does not exclude the possibility that other pathways may be involved. For example, Ca 2+ signaling/homeostasis are known to be responsible for the maintenance of cytoskeletal integrity (Tsai et al., 2015). Cholinergic activation of M 1 R coupled with the Gq/11 protein generates cytosolic calcium transients via phospholipase-C signaling pathway (Langmead et al., 2008). High Ca 2+ may act to increase the intrinsic GTP hydrolysis of tubulin and directly destabilize growing microtubule ends without changing the effective concentration of tubulin (O'Brien et al., 1997). Therefore, it is possible that excessive cholinergic signaling may imbalance intracellular Ca 2+ homeostasis and promotes tubulin destabilization. Antagonist FIGURE 10 | Model explaining the effect of M 1 R overexpression and antagonism on tubulin associated cytoskeleton and mitochondrial trafficking. (Left) M 1 R overexpressing neurons have increased basal activity in response to secreted acetylcholine signaling through overexpressed receptor. This is turn causes recruitment of trimeric G proteins that destabilize tubulin polymers by increasing intrinsic GTPase activity of tubulin. The lack of tubulin cytoskeleton in M 1 R overexpressing neuron leads to decreased mitochondrial trafficking and stagnation of mitochondria in the neurites that impairs outgrowth. (Right) M 1 R overexpression in the presence of antagonists MT7 and pirenzepine (PZ) stabilizes tubulin polymerization. The antagonists bind to the M 1 R and may stabilize a specific structural ensemble, which in turn recruit trimeric G proteins. However, the antagonist mediated M 1 R structural ensemble may sequester the bound G proteins and makes them unavailable for exerting their effect on tubulin polymerization which in turn stabilizes microtubule cytoskeleton and promotes mitochondrial trafficking and neurite outgrowth. mediated sequestration of G-proteins may limit this response and protect Ca 2+ induced tubulin destabilization. However, further experimentation is required to prove this hypothesis. Insertion and site-directed mutagenesis based studies have revealed potential G-protein interaction sites in the i2 and i3 loops in mAChRs (Blin et al., 1995;Liu et al., 1996;Hu et al., 2010). Overexpression of mutated M 1 R for disruptive Gα13 binding and subsequent reversal of cholinergic tubulin destabilization effect would provide another means of testing. The exact binding site for Gα13 protein in M 1 R needs to be evaluated.
Overexpression of M 1 R restricted the number of active mitochondria in neurites, presumably through diminished trafficking as a direct consequence of overexpressed M 1 Rinduced disruption of the cytoskeleton. The M 1 R antagonists, pirenzepine and MT7, were able to protect mitochondrial transport. Pirenzepine and MT7 also enhance mitochondrial oxygen consumption rate and respiratory complex activities through activation of the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α signaling axis (Calcutt et al., 2017). Mitochondrial function was also enhanced in sensory neuron cultures derived from M 1 R null mice. Thus, ACh signaling through M 1 R can negatively regulate mitochondrial phenotype at multiple levels in the neuron that include trafficking and positioning as well as fine regulation of activity of the respiratory complexes. Optimal regulation of mitochondrial function is critical in distal regions of sensory neurons which, in human peripheral nerve, can be up to a meter from the cell body (Schwarz, 2013). The architecture of sensory neurons poses an extreme cellular environment for mitochondrial distribution and a need to supply energy to the distal endings where energy demand is high (Bernstein and Bamburg, 2003;Chowdhury et al., 2013). Any defect in mitochondrial function is likely to have a profound influence on the axon. Recent in vivo studies of mitochondrial transport along the saphenous nerve of adult mice revealed elevated anterograde transport of this organelle in axons undergoing high rates of depolarization and impulse conduction (Sajic et al., 2013). Indeed, loss of function of mitochondrial proteins such as bcl-w or mitofusin-2 results in a length-dependent dying-back sensory neuropathy (Baloh et al., 2007;Misko et al., 2010;Courchesne et al., 2011). Genetic ablation of mitochondrial transport also leads to axonal growth failure following axotomy in mice (Zhou et al., 2016). In axons, approximately 30-40% of total mitochondria are constantly engaged in saltatory motion (Lovas and Wang, 2013). It is plausible that the microtubule disruption induced by overexpression of M 1 R eliminated the basic framework for motor proteins to carry their mitochondrial cargo and potentially other vesicular cargos. As a result, the physical abundance of active mitochondria was diminished in the distal neuritis, which would be expected to deprive the neuronal growth cone of an essential supply of ATP, resulting in suppressed actin treadmilling and neurite outgrowth. This hypothesis, summarized in Figure 10, is further supported by reports that several microtubule-targeted chemotherapeutic agents, such as colchicine and vincristine, are known to induce a sensory neuropathy in which the distal aspect of the sensory axon gradually degenerates (Bennett et al., 2014).
Our pre-clinical studies have demonstrated that selective and specific M 1 R antagonists promote neurite outgrowth in adult sensory neurons in vitro (Calcutt et al., 2017). MT7 and pirenzepine were also able to prevent or reverse the distal degenerative neuropathy characteristic of diabetes, chemotherapy-induced and HIV-induced peripheral neuropathy. In the present study, we have reinforced the finding that M 1 R antagonists are neuronal growth promoters using an unbiased automated high throughput neurite outgrowth measurement technique in a large cohort of sensory neurons. In addition, we have highlighted a molecular mechanism that indicates that treatment with these drugs stabilizes microtubules by sequestering Gα13 proteins and promotes microtubule-based axonal transport of mitochondria, which in turn augments neurite outgrowth. Peripheral neuropathy is a major cause of human morbidity with huge associated health care costs (Gordois et al., 2003;McInnes, 2012). One particularly encouraging implication of our identification of the endogenous M 1 Rmediated suppression of axonal outgrowth in sensory neurons is that antimuscarinic drugs that can prevent/reverse this process have already been widely used as approved drugs for other indications (Siatkowski et al., 2008). Since small fiber axonal degeneration is an early feature of many peripheral neuropathies, the novel growth-regulating pathway we have identified could be mobilized to prevent or reverse distal neurodegeneration. Our studies support a previously unrecognized therapeutic potential for M 1 R antagonists in the treatment of peripheral neuropathies and unravels a novel pathway of cholinergic signaling mediated via control of microtubule dynamics through Gα13 signaling.

AUTHOR CONTRIBUTIONS
MS conceptualized, designed and performed all the experiments, analyzed all data, prepared all figures, and wrote the manuscript. PF and NC obtained funding to support the work, aided in the design of experiments, and contributed to writing and editing of the manuscript. FUNDING type and Gα12/13 null HEK293 cell. (B) Immunoblots showing the relative amount of polymerized tubulin in wild type and Gα12/13 null HEK293 cells. (C) In BN-PAGE based assay, polymerized tubulin was stabilized in microtubule stabilization buffer and resolved in native gradient gel. The polymerized tubulin microtubules are trapped in the native page at the top (∼1200 kDa) whereas the dimers and monomers migrated to lower molecular weight regions. The first dimension gel was denatured and the proteins were further separated in 2nd dimension SDS-PAGE and immunoblotted. The spots at ∼146 and ∼66 kDa represent α-tubulin dimer and monomers. The red dotted circle represents the polymerized tubulin. Green and blue arrows indicate direction of protein movement. (D) Scatter plot showing polymerized tubulin in wild type and Gα12/13 null HEK293 cell as revealed by polymerized tubulin and BN-Page based assays. In the BN-PAGE assay the polymerized tubulin was quantified based on the relative intensity of the monomeric tubulin. N = 3/4 independent experiments. Data represented as mean ± SEM, p-value by unpaired t-test. (E) Immunofluorescent images showing actin (phalloidin) and (α-tubulin) in HEK293 (Wild type: WT) and GNAS12/13 knockout ( GNAS12/13) cells. Scale bar: 20 µm.