Changes in neuronal function that occur with age are an area of increasing importance. A potential significant contributor to age-dependent decline may be alterations to neurotransmitter release. Protein kinases, such as Protein Kinase C and Protein Kinase A, are well characterised modulators of neuronal function and neurotransmission. Protein Kinase D (PRKD) is a serine/threonine kinase whose role in neurons is less well characterised. Here we report that mutations in the C. elegans PRKD homolog, dkf-1 , show an acceleration in age-dependent decline of locomotion rate and an alteration to age-dependent changes in aldicarb sensitivity. These effects could be explained by a pre- or post-synaptic function of the protein kinase as the animal ages

Barclay, Jeff W

Johnson, James R

University of Liverpool Repository

 C. elegans dkf-1 (Protein Kinase D1) mutants have age-dependent defectsin locomotion and neuromuscular transmissionJames R. Johnson1, Jeff W. Barclay1§1Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, England, United Kingdom§To whom correspondence should be addressed: barclayj@liverpool.ac.ukAbstractChanges in neuronal function that occur with age are an area of increasing importance. A potential significant contributor toage-dependent decline may be alterations to neurotransmitter release. Protein kinases, such as Protein Kinase C and ProteinKinase A, are well characterised modulators of neuronal function and neurotransmission. Protein Kinase D (PRKD) is aserine/threonine kinase whose role in neurons is less well characterised. Here we report that mutations in the C. elegans PRKDhomolog, dkf-1, show an acceleration in age-dependent decline of locomotion rate and an alteration to age-dependent changesin aldicarb sensitivity. These effects could be explained by a pre- or post-synaptic function of the protein kinase as the animalages.Figure 1. Phenotypic analysis of dkf-1 mutants. :A. Thrashing rates of young (day 1 adulthood) Bristol N2 wild-types versus two independent dkf-1 mutants (ok2696, tm4906).Only the tm4906 strain showed a reduction in thrashing. B. Thrashing rates of older (day 5 adulthood) worms of the samestrains. Both dkf-1 strains showed a reduction in thrashing. C. Aldicarb sensitivity of young (day 1) Bristol N2 wild-typesversus two independent dkf-1 mutants. Both dkf-1 strains were hypersensitive to aldicarb in comparison to wild-types D.Aldicarb sensitivity of older (day 5) worms of the same strains. Both dkf-1 strains were resistant to aldicarb in comparison towild-types. 4/4/2023 - Open Access DescriptionProtein Kinase D1 (PRKD1) is a serine/threonine protein kinase associated with numerous cellular phenotypes including cellproliferation and survival, oxidative stress, trafficking and immunity (Zhang et al., 2021). As such, defects in PRKD1 lead tocancer, kidney disease, cardiovascular disease and inflammatory disease. PRKD1 is also expressed in neurons where it playsroles in development, survival, polarity and pain transmission (Li and Wang, 2014).In addition to a serine/threonine kinase domain, structurally PRKD1 contains two upstream C1 domains that are thought to beactivated by the lipid second messenger diacylglycerol (DAG) where it recruits the enzyme to the plasma membrane, in asimilar manner to protein kinase C (PKC) (Zhang et al., 2021). In addition to DAG, PRKD1 also appears to requiretransphosphorylation by PKC to become activated. Once activated PRKD1 phosphorylates a host of downstream proteins(Franz-Wachtel et al., 2012).Both of these upstream activation factors, DAG and PKC, are well known to alter synaptic signalling. DAG altersneurotransmitter release via direct interaction with the synaptic priming protein unc-13/Munc13 (Lackner et al., 1999, Rhee etal., 2002) and indirectly through the activation of PKC. In terms of synaptic signalling, the main characterised targets for PKCphosphorylation include SNAP-25 and Stxbp1/Munc18 (Barclay et al., 2003, Barclay et al., 2005). Despite its neuronalexpression and its function downstream of both DAG and PKC, very little is known connecting PRKD1 to synaptic signalling.In Caenorhabditis elegans, genetic mutations that increase levels of DAG or the addition of a pharmacological DAG analogue,the phorbol ester PMA, increase locomotion rate and make worms hypersensitive to aldicarb (Lackner et al., 1999, Nurrish etal., 1999). Conversely, loss-of-function mutations that block the downstream action of DAG or PMA, such as PKC (pkc-1,pkc-2), or expression of DAG-insensitive unc-13/Munc13 make worms resistant to the effects of aldicarb (Lackner et al.,1999, Sieburth et al., 2007, Edwards et al., 2012). Our understanding of the effects of PRKD1 on locomotion in C. elegans islimited and we know nothing of its effects on aldicarb strength.In C. elegans, there are two PRKD1 isoforms, the best conserved of which is dkf-1. dkf-1 is expressed in neurons around thenematode pharynx as well as in some tail neurons (Feng et al., 2006) whereas the other nematode isoform, dkf-2, is alsoneuronally expressed and contributes to lifespan and stress responses (Feng et al., 2007). Previously a locomotion defect hasbeen reported for a dkf-1 genetic mutation and following RNAi knockdown targeting dkf-1 (Feng et al., 2006); however, theextent of locomotion defect was not explicitly quantified.We therefore measured the locomotion rate directly by assessing thrashing in liquid of two dkf-1 mutant alleles in comparisonto Bristol N2 wild-type worms (Figure 1A). In contrast with previous reports (Feng et al., 2006), we found only marginaleffects of the dkf-1 mutations on locomotion rate when assessed at a young age (day 1 of adulthood – Figure 1A). The dkf-1ok2696 mutation had no effect on rate of thrashing, whereas the tm4906 allele exhibited a small, but significant (~12%)decrease. A strong defect in locomotion, however, became evident with age. By day 5 of adulthood, an age-dependentdecrease in locomotion for the Bristol N2 wild-type was minimal; however, there was a substantial, significant decrease inlocomotion for both of the dkf-1 mutant alleles (Figure 1B). The ok2696 and the tm4906 alleles had 96% and 81% age-dependent reductions in thrashing in comparison to only 13% for Bristol N2 wild-types.C. elegans locomotion is impacted by effects on many different physiological properties, including neuromuscular signallingstrength. Therefore, we assessed neurotransmission for our strains by aldicarb assay (Mahoney et al., 2006). Unlike mutationsof PKC that cause worms to be resistant to inhibitors of cholinesterase (Sieburth et al., 2007, Edwards et al., 2012),surprisingly we found that either of the dkf-1 mutations caused hypersensitivity to aldicarb at an early age (Figure 1C).Aldicarb sensitivity is known to increase with age in worms (Mulcahy et al., 2013) and we confirmed that we could quantifyan increase in sensitivity for Bristol N2 at day 5 (Figure 1D). Strikingly, by day 5 both of the dkf-1 mutant alleles demonstratedthe opposite age-dependent effect to wild-types, becoming more resistant to aldicarb (Figure 1D).Our results indicate that mutations in the PRKD1 orthologue dkf-1 are associated with hypersensitivity to aldicarb at a youngage, which is the opposite effect to that seen for PKC isoforms. This may be the result of an increase in endogenousneurotransmitter release. PRKD isoforms have been shown to phosphorylate a number of proteins that could potentiallyimpact exocytosis, including those involved in post-Golgi vesicle trafficking and the cytoskeleton (Eisler et al., 2018, Weeberet al., 2019, Loza-Valdes et al., 2021, Grimaldi et al., 2022). Alternatively, although expression of dkf-1 was not previouslyshown in body wall muscles, PRKD1 is expressed in muscle in humans where it interacts with the muscle protein titin (Herwiget al., 2020). Therefore, it is possible that dkf-1 is also expressed in body wall muscle in C. elegans and that this expressioncontributes to a postsynaptic impact on aldicarb sensitivity. In support of this idea, overexpression of dkf-1 did cause adecrease in nematode body length (Feng et al., 2006).We have also identified some interesting age-dependent effects of dkf-1 mutation. The almost complete reduction in thrashingrate and an inversion of aldicarb phenotype by day 5 of adulthood supports a progressive function for dkf-1. Indeed, PRKD1 is 4/4/2023 - Open Access linked to neurodegeneration via a role in oxidative stress (Ay et al., 2015, Pose-Utrilla et al., 2017) and dkf-2 worms have analtered lifespan (Feng et al., 2007).Nematode locomotion rate is known to decrease with age and specific mutations, such ascysteine string protein / dnj-14, can accelerate this decline (Kashyap et al., 2014). Mutations in many genes can alter aldicarbsensitivity in nematodes (Sieburth et al., 2005) and that sensitivity normally increases with age (Mulcahy et al., 2013). Theswitch from aldicarb hypersensitivity to resistance evident in dkf-1 mutants implies a potential progressive effect onneuromuscular signalling or postsynaptic contraction during ageing. Future research will be essential to explore such age-dependent mechanisms in whole-animal models in more detail.MethodsC. elegans strains: All strains were cultured under standard conditions on Nematode Growth Medium agar plates at 20°C withEscherichia coli OP50 as a food source (Brenner, 1974). The following strains were used in this study: Bristol N2 (wild-type),RB2038 and FX04906. Both mutants have small (~400-500bp) N-terminal deletions of the dkf-1 gene and were notbackcrossed into Bristol N2 before phenotypic analysis. Only phenotypes observed in both independently derived alleles wereconsidered specific to dkf-1 function.Phenotypic assays: All assays were performed on adult hermaphrodites at 20°C in a temperature-controlled room. Agesynchronisation of worms was achieved by bleaching. During ageing, worms were picked to fresh Nematode Growth Mediumagar plates daily to maintain age synchronisation of the worm population. Locomotion rate was quantified by thrashing inDent’s solution (140 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4 with bovine serum albumin at0.1 mg/ml) as described previously (Johnson et al., 2016, Johnson et al., 2017). One thrash was defined as one completemovement from maximum to minimum amplitude and back. Acute sensitivity to aldicarb was quantified by assessing the timeto paralysis of a population of worms (Mahoney et al., 2006, Edwards et al., 2012). For each experiment, worms were placedon an unseeded Nematode Growth Medium agar plate supplemented with 1 mM aldicarb. Paralysis was determined by afailure to respond to a light mechanical stimulation assessed every 10 minutes following exposure to aldicarb.Statistical analysis: All data are expressed as mean ± S.E. 20 worms per strain per age were used to assess locomotion.Significance for thrashing was assessed by one-way analysis of variance (ANOVA) with Tukey post-hoc test for multiplecomparisons. For each aldicarb experiment, 25-30 worms per strain were used. Significance to aldicarb sensitivity wasassessed by two-way analysis of variance (ANOVA) with Tukey post-hoc test for multiple comparisons. Experiments wereperformed three times.ReagentsStrain Genotype Available fromBristol N2 Wild-type C. elegans CGCRB2038 dkf-1 (ok2696) CGCFX04906 dkf-1 (tm4906) Mitani labAcknowledgements: Strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by theNational Institutes of Health National Center for Research Resources, and by the National Bioresource Project for theExperimental Animal “Nematode C. elegans.ReferencesAy M, Jin H, Harischandra DS, Asaithambi A, Kanthasamy A, Anantharam V, Kanthasamy AG. 2015. Molecular cloning,epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models ofParkinson's disease. J Neurochem 135: 402-15. PubMed ID: 26230914Barclay JW, Craig TJ, Fisher RJ, Ciufo LF, Evans GJ, Morgan A, Burgoyne RD. 2003. Phosphorylation of Munc18 by proteinkinase C regulates the kinetics of exocytosis. J Biol Chem 278: 10538-45. PubMed ID: 12519779Barclay JW, Morgan A, Burgoyne RD. 2005. Calcium-dependent regulation of exocytosis. Cell Calcium 38: 343-53. PubMedID: 16099500 4/4/2023 - Open Access Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94. PubMed ID: 4366476Edwards MR, Johnson JR, Rankin K, Jenkins RE, Maguire C, Morgan A, Burgoyne RD, Barclay JW. 2012. PKC-2phosphorylation of UNC-18 Ser322 in AFD neurons regulates temperature dependency of locomotion. J Neurosci 32: 7042-51. PubMed ID: 22593072Eisler SA, Curado F, Link G, Schulz S, Noack M, Steinke M, Olayioye MA, Hausser A. 2018. A Rho signaling network linksmicrotubules to PKD controlled carrier transport to focal adhesions. Elife 7: . PubMed ID: 30028295Feng H, Ren M, Chen L, Rubin CS. 2007. Properties, regulation, and in vivo functions of a novel protein kinase D:Caenorhabditis elegans DKF-2 links diacylglycerol second messenger to the regulation of stress responses and life span. J BiolChem 282: 31273-88. PubMed ID: 17728253Feng H, Ren M, Wu SL, Hall DH, Rubin CS. 2006. Characterization of a novel protein kinase D: Caenorhabditis elegansDKF-1 is activated by translocation-phosphorylation and regulates movement and growth in vivo. J Biol Chem 281: 17801-14.PubMed ID: 16613841Franz-Wachtel M, Eisler SA, Krug K, Wahl S, Carpy A, Nordheim A, et al., Macek B. 2012. Global detection of proteinkinase D-dependent phosphorylation events in nocodazole-treated human cells. Mol Cell Proteomics 11: 160-70. PubMed ID:22496350Grimaldi G, Filograna A, Schembri L, Lo Monte M, Di Martino R, Pirozzi M, et al., Corda D. 2022. PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane. ProcNatl Acad Sci U S A 119: . PubMed ID: 34969853Herwig M, Kolijn D, Lódi M, Hölper S, Kovács Á, Papp Z, et al., Hamdani N. 2020. Modulation of Titin-Based Stiffness inHypertrophic Cardiomyopathy via Protein Kinase D. Front Physiol 11: 240. PubMed ID: 32351396Johnson JR, Edwards MR, Davies H, Newman D, Holden W, Jenkins RE, et al., Barclay JW. 2017. Ethanol StimulatesLocomotion via a G(αs)-Signaling Pathway in IL2 Neurons in Caenorhabditis elegans. Genetics 207: 1023-1039. PubMed ID:28951527Johnson JR, Rajamanoharan D, McCue HV, Rankin K, Barclay JW. 2016. Small Heat Shock Proteins Are Novel CommonDeterminants of Alcohol and Nicotine Sensitivity in Caenorhabditis elegans. Genetics 202: 1013-27. PubMed ID: 26773049Kashyap SS, Johnson JR, McCue HV, Chen X, Edmonds MJ, Ayala M, et al., Morgan A. 2014. Caenorhabditis elegans dnj-14,the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform forneuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol. Hum Mol Genet 23: 5916-27.PubMed ID: 24947438Lackner MR, Nurrish SJ, Kaplan JM. 1999. Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta:DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24: 335-46. PubMed ID: 10571228Li G, Wang Y. 2014. Protein kinase D: a new player among the signaling proteins that regulate functions in the nervoussystem. Neurosci Bull 30: 497-504. PubMed ID: 24526660Loza-Valdes A, Mayer AE, Kassouf T, Trujillo-Viera J, Schmitz W, Dziaczkowski F, et al., Sumara G. 2021. Aphosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism. Life Sci Alliance 4:. PubMed ID: 34145024Mahoney TR, Luo S, Nonet ML. 2006. Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat Protoc 1: 1772-7. PubMed ID: 17487159Mulcahy B, Holden-Dye L, O'Connor V. 2013. Pharmacological assays reveal age-related changes in synaptic transmission atthe Caenorhabditis elegans neuromuscular junction that are modified by reduced insulin signalling. J Exp Biol 216: 492-501.PubMed ID: 23038730Nurrish S, Ségalat L, Kaplan JM. 1999. Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance ofUNC-13 at release sites. Neuron 24: 231-42. PubMed ID: 10677040Pose-Utrilla J, García-Guerra L, Del Puerto A, Martín A, Jurado-Arjona J, De León-Reyes NS, et al., Iglesias T. 2017.Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1. NatCommun 8: 2275. PubMed ID: 29273751Rhee JS, Betz A, Pyott S, Reim K, Varoqueaux F, Augustin I, et al., Brose N. 2002. Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108: 121-33. PubMed ID: 4/4/2023 - Open Access 11792326Sieburth D, Ch'ng Q, Dybbs M, Tavazoie M, Kennedy S, Wang D, et al., Kaplan JM. 2005. Systematic analysis of genesrequired for synapse structure and function. Nature 436: 510-7. PubMed ID: 16049479Sieburth D, Madison JM, Kaplan JM. 2007. PKC-1 regulates secretion of neuropeptides. Nat Neurosci 10: 49-57. PubMed ID:17128266Weeber F, Becher A, Seibold T, Seufferlein T, Eiseler T. 2019. Concerted regulation of actin polymerization duringconstitutive secretion by cortactin and PKD2. J Cell Sci 132: . PubMed ID: 31727638Zhang X, Connelly J, Chao Y, Wang QJ. 2021. Multifaceted Functions of Protein Kinase D in Pathological Processes andHuman Diseases. Biomolecules 11: . PubMed ID: 33807058Funding: Wellcome TrustAuthor Contributions: James R. Johnson: investigation, methodology, formal analysis. Jeff W. Barclay: conceptualization,writing - review editing.Reviewed By: AnonymousHistory: Received March 13, 2023 Revision Received March 24, 2023 Accepted April 4, 2023 Published Online April 4,2023 Indexed April 18, 2023Copyright: © 2023 by the authors. This is an open-access article distributed under the terms of the Creative CommonsAttribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original author and source are credited.Citation: Johnson, JR; Barclay, JW (2023). C. elegans dkf-1 (Protein Kinase D1) mutants have age-dependent defects inlocomotion and neuromuscular transmission. microPublication Biology. 10.17912/micropub.biology.000800 4/4/2023 - Open Access

<i>C. elegans dkf-1</i> (Protein Kinase D1) mutants have age-dependent defects in locomotion and neuromuscular transmission.

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<i>C. elegans dkf-1</i> (Protein Kinase D1) mutants have age-dependent defects in locomotion and neuromuscular transmission.

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