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• W2550519436 abstract "The γ2 subunit of GABA type A receptors (GABAARs) is thought to be subject to palmitoylation by both Golgi-associated DHHC-type zinc finger protein (GODZ; also known as DHHC3) and its paralog Sertoli cell gene with a zinc finger domain-β (SERZ-β; DHHC7) based on overexpression of enzymes and substrates in heterologous cells. Here we have further investigated the substrate specificity of these enzymes by characterization of GODZ and SERZ-β knock-out (KO) mice as well as double KO (DKO) neurons. Palmitoylation of γ2 and a second substrate, growth-associated protein of 43 kDa, that is independently implicated in trafficking of GABAARs was significantly reduced in brain of GODZ KO versus wild-type (WT) mice but unaltered in SERZ-β KO mice. Accumulation of GABAARs at synapses, GABAergic innervation, and synaptic function were reduced in GODZ KO and DKO neurons to a similar extent, indicating that SERZ-β does not contribute to palmitoylation or trafficking of GABAARs even in the absence of GODZ. Notably, these effects were seen only when mutant neurons were grown in competition with WT neurons, thereby mimicking conditions of shRNA-transfected neurons previously used to characterize GODZ. However, GABA-evoked whole-cell currents of DKO neurons and the GABAAR cell surface expression in DKO neurons and GODZ or SERZ-β KO brain slices were unaltered, indicating that GODZ-mediated palmitoylation selectively controls the pool of receptors at synapses. The different substrate specificities of GODZ and SERZ-β in vivo were correlated with their differential localization to cis- versus trans-Golgi compartment, a mechanism that was compromised by overexpression of GODZ. The γ2 subunit of GABA type A receptors (GABAARs) is thought to be subject to palmitoylation by both Golgi-associated DHHC-type zinc finger protein (GODZ; also known as DHHC3) and its paralog Sertoli cell gene with a zinc finger domain-β (SERZ-β; DHHC7) based on overexpression of enzymes and substrates in heterologous cells. Here we have further investigated the substrate specificity of these enzymes by characterization of GODZ and SERZ-β knock-out (KO) mice as well as double KO (DKO) neurons. Palmitoylation of γ2 and a second substrate, growth-associated protein of 43 kDa, that is independently implicated in trafficking of GABAARs was significantly reduced in brain of GODZ KO versus wild-type (WT) mice but unaltered in SERZ-β KO mice. Accumulation of GABAARs at synapses, GABAergic innervation, and synaptic function were reduced in GODZ KO and DKO neurons to a similar extent, indicating that SERZ-β does not contribute to palmitoylation or trafficking of GABAARs even in the absence of GODZ. Notably, these effects were seen only when mutant neurons were grown in competition with WT neurons, thereby mimicking conditions of shRNA-transfected neurons previously used to characterize GODZ. However, GABA-evoked whole-cell currents of DKO neurons and the GABAAR cell surface expression in DKO neurons and GODZ or SERZ-β KO brain slices were unaltered, indicating that GODZ-mediated palmitoylation selectively controls the pool of receptors at synapses. The different substrate specificities of GODZ and SERZ-β in vivo were correlated with their differential localization to cis- versus trans-Golgi compartment, a mechanism that was compromised by overexpression of GODZ. S-Palmitoylation is an important posttranslational modification that involves the addition of the 16-carbon fatty acid chain palmitate via a thioester bond to Cys residues (1.Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (432) Google Scholar). This modification in turn can alter a protein's conformational state, membrane association, and complex formation as well as its susceptibility to other posttranslational modifications (2.Greaves J. Chamberlain L.H. Palmitoylation-dependent protein sorting.J. Cell Biol. 2007; 176: 249-254Crossref PubMed Scopus (193) Google Scholar, 3.Salaun C. Greaves J. Chamberlain L.H. The intracellular dynamic of protein palmitoylation.J. Cell Biol. 2010; 191: 1229-1238Crossref PubMed Scopus (233) Google Scholar4.Blaskovic S. Blanc M. van der Goot F.G. What does S-palmitoylation do to membrane proteins?.FEBS J. 2013; 280: 2766-2774Crossref PubMed Scopus (164) Google Scholar). Global analyses of rat synaptosomal fractions led to the discovery of nearly 300 palmitoylated synaptosomal protein candidates that illustrate the particular importance of palmitoylation in regulating the function of neuronal synapses (5.Kang R. Wan J. Arstikaitis P. Takahashi H. Huang K. Bailey A.O. Thompson J.X. Roth A.F. Drisdel R.C. Mastro R. Green W.N. Yates 3rd, J.R. Davis N.G. El-Husseini A. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.Nature. 2008; 456: 904-909Crossref PubMed Scopus (419) Google Scholar). In mammalian cells, the palmitoylation reaction is catalyzed principally by a super gene family of 23 palmitoyl acyltransferases containing a DHHC motif in a cysteine-rich domain (DHHC-CRD) 3The abbreviations used are: DHHC-CRD, DHHC motif in a cysteine-rich domain; GABAAR, GABA type A receptor; GODZ, Golgi-associated DHHCtype zinc finger protein; SERZ-⁁, Sertoli cell gene with a zinc finger domain-⁁; DKO, double KO; GAP-43, growth-associated protein of 43 kDa; PAT, palmitoyl acyltransferase; PSD, postsynaptic density; AMPAR, AMPA receptor; DIV, days in vitro; GAD, glutamic acid decarboxylase; n.s., nonsignificant; ANOVA, analysis of variance; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; ABE, acyl-biotin exchange; GalT, galactosyltransferase; SIM, structured illumination microscopy; ICQ, intensity correlation quotient; PGK-neo, phosphoglycerate kinase-neomycin resistance gene cassette; BAC, bacterial artificial chromosome; NHS, N-hydroxysuccinimide; ACSF, artificial cerebrospinal fluid; NA, numerical aperture. that are both essential for enzyme function (6.Fukata Y. Fukata M. Protein palmitoylation in neuronal development and synaptic plasticity.Nat. Rev. Neurosci. 2010; 11: 161-175Crossref PubMed Scopus (426) Google Scholar7.Greaves J. Chamberlain L.H. DHHC palmitoyl transferases: substrate interactions and (patho)physiology.Trends Biochem. Sci. 2011; 36: 245-253Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 8.Korycka J. Łach A. Heger E. Bogusławska D.M. Wolny M. Toporkiewicz M. Augoff K. Korzeniewski J. Sikorski A.F. Human DHHC proteins: a spotlight on the hidden player of palmitoylation.Eur. J. Cell Biol. 2012; 91: 107-117Crossref PubMed Scopus (97) Google Scholar, 9.Jennings B.C. Linder M.E. DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.J. Biol. Chem. 2012; 287: 7236-7245Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar10.Gottlieb C.D. Zhang S. Linder M.E. The cysteine-rich domain of the DHHC3 palmitoyltransferase is palmitoylated and contains tightly bound zinc.J. Biol. Chem. 2015; 290: 29259-29269Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The mechanisms that determine substrate specificity of PATs remain poorly understood, although some specificity is observed upon overexpression of substrates and PATs in heterologous cells. However, increasing evidence suggests that the substrate specificity of DHHC-type PATs in vivo is much more stringent than in vitro. For example, the postsynaptic density (PSD) protein of 95-kDa (PSD-95) can be palmitoylated in heterologous cells by at least five members of the DHHC family of PATs (11.Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar), whereas in vivo only DHHC8 and DHHC2 have so far been shown to be required for normal palmitoylation of PSD-95 (12.Ho G.P. Selvakumar B. Mukai J. Hester L.D. Wang Y. Gogos J.A. Snyder S.H. S-Nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD-95.Neuron. 2011; 71: 131-141Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 13.Fukata Y. Dimitrov A. Boncompain G. Vielemeyer O. Perez F. Fukata M. Local palmitoylation cycles define activity-regulated postsynaptic subdomains.J. Cell Biol. 2013; 202: 145-161Crossref PubMed Scopus (181) Google Scholar). In part, the substrate specificity of DHHC enzymes may be determined by their segregation to different subcellular compartments, which has also implications as to how palmitoylation affects the trafficking or function of substrate proteins (14.Ohno Y. Kihara A. Sano T. Igarashi Y. Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.Biochim. Biophys. Acta. 2006; 1761: 474-483Crossref PubMed Scopus (319) Google Scholar). Here we have extended our characterization of GODZ (also known as DHHC3) and its most closely related paralog, Sertoli cell gene with a zinc finger domain-β (SERZ-β; DHHC7), which are known as founding members of the mammalian DHHC family of proteins (15.Uemura T. Mori H. Mishina M. Isolation and characterization of Golgi apparatus-specific GODZ with the DHHC zinc finger domain.Biochem. Biophys. Res. Commun. 2002; 296: 492-496Crossref PubMed Scopus (50) Google Scholar, 16.Chaudhary J. Skinner M.K. Identification of a novel gene product, Sertoli cell gene with a zinc finger domain, that is important for FSH activation of testicular Sertoli cells.Endocrinology. 2002; 143: 426-435Crossref PubMed Scopus (22) Google Scholar). We have previously provided evidence that in cultured neurons GODZ is important as a PAT of γ2 subunit-containing γ-aminobutyric acid (GABA) type A receptors (GABAARs), the principal receptors that mediate GABAergic synaptic inhibition (17.Keller C.A. Yuan X. Panzanelli P. Martin M.L. Alldred M. Sassoè-Pognetto M. Lüscher B. The γ2 subunit of GABAA receptors is a substrate for palmitoylation by GODZ.J. Neurosci. 2004; 24: 5881-5891Crossref PubMed Scopus (200) Google Scholar). Moreover, GODZ shRNA- or dominant negative GODZ-mediated knockdown of GODZ suggested that GODZ functions selectively at inhibitory but not excitatory synapses (18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar). Nevertheless, GODZ has independently been proposed to also regulate the trafficking of ionotropic glutamate receptors as both AMPA and NMDA receptors can be palmitoylated upon overexpression of GODZ in heterologous cells and neurons (19.Hayashi T. Rumbaugh G. Huganir R.L. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites.Neuron. 2005; 47: 709-723Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 20.Hayashi T. Thomas G.M. Huganir R.L. Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking.Neuron. 2009; 64: 213-226Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Based on mRNA expression analyses, GODZ and SERZ-β show similar tissue-specific expression in vivo and in brain are selectively expressed in neurons (18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar). When examined by overexpression in heterologous cells, both enzymes exhibit broad substrate specificity, and indeed virtually any palmitoylated protein can be palmitoylated by both GODZ and SERZ-β under these conditions (11.Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar, 21.Oku S. Takahashi N. Fukata Y. Fukata M. In silico screening for palmitoyl substrates reveals a role for DHHC1/3/10 (zDHHC1/3/11)-mediated neurochondrin palmitoylation in its targeting to Rab5-positive endosomes.J. Biol. Chem. 2013; 288: 19816-19829Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, the extent by which any of these putative substrate proteins are palmitoylated by GODZ or SERZ-β in vivo has not been addressed. To begin to address the role GODZ-mediated palmitoylation plays by loss of function analyses in vivo, here we have generated GODZ and SERZ-β knock-out (KO) mice. Analyses of GODZ KO and GODZ/SERZ-β double KO (DKO) primary cultured neurons confirmed that GODZ is important for normal accumulation of GABAARs at synapses and receptor-dependent presynaptic innervation. However, this phenotype was only observed when mutant neurons were grown and analyzed under conditions where they were forced to compete with wild-type (WT) neurons for GABAergic innervation, i.e. conditions we previously showed to be highly sensitive to reduced expression of the γ2 subunit of GABAARs (22.Ren Z. Sahir N. Murakami S. Luellen B.A. Earnheart J.C. Lal R. Kim J.Y. Song H. Luscher B. Defects in dendrite and spine maturation and synaptogenesis associated with an anxious-depressive-like phenotype of GABAA receptor-deficient mice.Neuropharmacology. 2015; 88: 171-179Crossref PubMed Scopus (29) Google Scholar). By contrast, the cell surface expression and synaptic localization of GABAARs and the density of GABAergic synapses were unaffected in pure DKO cultures. Nevertheless, palmitoylation of γ2 was markedly reduced in brain of GODZ KO compared with WT mice along with reduced palmitoylation of growth-associated protein of 43 kDa (GAP-43) (also known as neuromodulin) as a second substrate that was recently implicated in the formation of GABAergic synapses (23.Wang C.Y. Lin H.C. Song Y.P. Hsu Y.T. Lin S.Y. Hsu P.C. Lin C.H. Hung C.C. Hsu M.C. Kuo Y.M. Lee Y.J. Hsu C.Y. Lee Y.H. Protein kinase C-dependent growth-associated protein 43 phosphorylation regulates gephyrin aggregation at developing GABAergic synapses.Mol. Cell. Biol. 2015; 35: 1712-1726Crossref PubMed Scopus (17) Google Scholar). Consistent with all these findings, electrophysiological analyses of DKO neurons revealed functional defects that were principally limited to inhibitory synapses. Notably, palmitoylation of PSD-95 and AMPARs was unaltered in brain of GODZ and SERZ-β KO mice. Moreover, palmitoylation of γ2 and GAP-43 was unaltered in SERZ-β KO mice. The data indicate substantially greater substrate specificity of both PATs in vivo versus in vitro. Super-resolution microscopy showed that GODZ is highly restricted to the cis-Golgi, whereas SERZ-β was enriched in the trans-Golgi. Such differences in the subcellular localization of PATs might contribute to the increased substrate specificity of these enzymes in vivo versus after overexpression of substrate and enzyme in heterologous cells. To enable investigation of the role of GODZ- and SERZ-β-mediated palmitoylation by loss of function analyses in vivo, we generated KO mice in which the third and fourth protein-encoding exons of each of these two genes were deleted by homologous recombination in embryonic stem cells (Fig. 1, a and b, and “Experimental Procedures”). These two exons together encode the DHHC-CRD that is essential for enzyme function of GODZ and other DHHC family PATs (9.Jennings B.C. Linder M.E. DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.J. Biol. Chem. 2012; 287: 7236-7245Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 10.Gottlieb C.D. Zhang S. Linder M.E. The cysteine-rich domain of the DHHC3 palmitoyltransferase is palmitoylated and contains tightly bound zinc.J. Biol. Chem. 2015; 290: 29259-29269Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). For both GODZ and SERZ-β, deletion of exons 3 and 4 is predicted to cause a shift in the translational reading frame of the downstream exons with artificial stop sites and hence to result in enzymatically inactive, truncated, and likely unstable proteins that contain the protein sequence of only the first two of a total of six protein-coding exons. Successful deletion of exons 3 and 4 was verified by Southern blotting of genomic DNA using DNA probes that flanked the targeting vector (not shown) and by reverse transcription (RT)-PCR analyses of total brain RNA (Fig. 1c). For each gene, RT-PCR of RNA isolated from WT and heterozygous mice using primer pairs that annealed to exons 1 and 4 (GP1/2 and SP1/2, respectively; Fig. 1, a and b) revealed the expected RT-PCR amplification products in WT mice that were absent from corresponding KO mice as expected (Fig. 1c, upper micrographs, primer pairs GP1/2 and SP1/2). Moreover, PCR analyses of reverse transcribed RNA using primers that annealed to exons 1 and 6 of each gene (GP1/3 and SP1/3, respectively; Fig. 1, a and b) revealed the presence of the predicted cDNAs of WT mice and the predicted truncated products corresponding to cDNAs that had exons 3 and 4 deleted (Fig. 1c, lower micrographs, primer pairs GP1/3 and SP1/3). RNA isolated from heterozygous GODZ and SERZ-β mice gave rise to both the WT and truncated RT-PCR products as expected (Fig. 1c). Next we confirmed the absence of GODZ in KO mice by Western blotting (Fig. 1d, left panels). Immunoblotting of whole brain lysates of WT and SERZ-β KO mice probed for GODZ detected a ∼31-kDa protein corresponding in size to previously described epitope-tagged recombinant GODZ, which is slightly smaller than predicted based on its 299-amino acid sequence (17.Keller C.A. Yuan X. Panzanelli P. Martin M.L. Alldred M. Sassoè-Pognetto M. Lüscher B. The γ2 subunit of GABAA receptors is a substrate for palmitoylation by GODZ.J. Neurosci. 2004; 24: 5881-5891Crossref PubMed Scopus (200) Google Scholar). Importantly, this protein was absent in brain lysates of GODZ KO mice as expected (Fig. 1d, left panels). Immunoblots for heat shock protein of 90 kDa (HSP90) analyzed in parallel were used as loading controls (Fig. 1d, lower panels). Notably, the GODZ antiserum used for Western blotting was directed against a C-terminal 31-amino acid peptide of GODZ that is absent in the putative truncated protein of GODZ KO mice because of the deletion-induced, altered translational reading frame. Parallel analyses of the same brain lysates with a SERZ-β antiserum detected a protein with an Mr of ∼30,000, which again is slightly less than the predicted molecular weight for this 308-amino acid protein. As predicted, this protein was present in extracts of WT and GODZ KO mice and absent in SERZ-β KO mice (Fig. 1d, right panels). The SERZ-β antiserum used for these experiments is directed against a peptide containing amino acids 40–193 of human SERZ-β that includes a 37-amino acid exon that is absent in mouse SERZ-β, whereas the rest of the immunogen is 98% conserved between human and mouse. In addition to the expected ∼30-kDa SERZ-β protein species, this antiserum detected an additional, more slowly migrating SERZ-β-immunoreactive band (∼35 kDa) that was also absent in SERZ-β KO mice and therefore may be SERZ-β-related (Fig. 1d, right panels). It could represent a detergent-resistant complex of SERZ-β with another, as of yet unknown protein. Next we verified the absence of GODZ immunoreactivity from cultured GODZ KO neurons and brain sections using a GODZ antibody directed against the C-terminal 50-amino acid peptide of mouse GODZ. Immunostaining for the microtubule-associated protein MAP2 was used to illustrate the outlines of neurons and dendrites. Immunofluorescent staining of primary cultured cortical neurons at 18 days in vitro (DIV 18) confirmed the Golgi-specific staining of GODZ described previously (17.Keller C.A. Yuan X. Panzanelli P. Martin M.L. Alldred M. Sassoè-Pognetto M. Lüscher B. The γ2 subunit of GABAA receptors is a substrate for palmitoylation by GODZ.J. Neurosci. 2004; 24: 5881-5891Crossref PubMed Scopus (200) Google Scholar). Moreover, this staining was entirely absent in cultures prepared from GODZ KO embryos (Fig. 1e). In brain sections of WT mice, GODZ immunoreactivity was similarly delimited to Golgi-like structures of neurons as expected, and this staining was absent in sections of GODZ KO mice (Fig. 1f). Immunostaining for the inhibitory synapse marker gephyrin was used to visually delineate the neuropil from neuronal somata of brain sections. Notably, analogous immunostainings for SERZ-β were not feasible as the SERZ-β antiserum used for Western blotting revealed only unspecific staining under these (non-denaturing) conditions (not shown). Nevertheless, all our analyses were consistent with faithful and specific inactivation of the GODZ and SERZ-β genomic loci in the respective KO mice. Both GODZ KO and SERZ-β KO mice were found to be viable and fertile without overt behavioral or physical phenotypes. Nevertheless, male GODZ KO mice showed a modest reduction in body weight (89.2 ± 2.5% of WT at 1 month of age, p < 0.001; 90.4 ± 1.7% of WT at 2 months of age, p < 0.01; n = 14–18 for all groups, t tests). By contrast, the body weights of female GODZ KO mice and male and female SERZ-β KO mice were not measurably affected (not shown). Previous in vitro experiments using overexpression of recombinant proteins had indicated largely overlapping substrate specificity of GODZ and SERZ-β (Ref. 18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar; and for review, see Ref. 6.Fukata Y. Fukata M. Protein palmitoylation in neuronal development and synaptic plasticity.Nat. Rev. Neurosci. 2010; 11: 161-175Crossref PubMed Scopus (426) Google Scholar). Therefore, to test for possible functional redundancy of GODZ and SERZ-β, we interbred GODZ and SERZ-β KO mice to generate GODZ/SERZ-β DKO mice. Unexpectedly, most DKO mice showed a perinatally lethal phenotype, although early on during the course of these studies a few DKO mice survived to adulthood. By 1 year of age these surviving DKO mice showed drastically reduced body and brain weights when compared with littermate controls (total body weight of DKO mice, 53.6 ± 1.7% of GODZ+/− SERZ-β+/− and GODZ+/+ SERZ-β−/− controls; DKO brain weight, 74.7 ± 6.3% of controls; n = 3 mice/group). No viable postnatal DKO mice were recovered beyond three generations of brother-sister matings of GODZ+/− SERZ-β+/− mice. As predicted, these findings suggested at least partial functional redundancy of GODZ and SERZ-β and, by extension, significant overlap in substrate specificity of these two enzymes. In addition, they dictated that further analyses of the neuronal mutant phenotypes were limited to single KO mice or to cultured neurons derived from KO or DKO embryos. Given the defects in GABAAR clustering and synapse formation seen previously in shRNA- and dominant negative GODZ-transfected neurons (18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar), we predicted that such deficits should be replicated in neuron cultures prepared from GODZ KO neurons. Surprisingly, cortical neurons prepared from GODZ KO embryos (18 DIV) showed normal GABAAR clustering as evidenced by unaltered density of punctate immunostaining for the γ2 subunit and the presynaptic marker glutamic acid decarboxylase (GAD) in GODZ KO compared with WT cortical cultures (Fig. 2, a–d versus e–h, m, and n) (mean number of γ2 puncta/40 μm dendrite: WT, 17.5 ± 1.1, GODZ KO, 16.8 ± 0.9; p, n.s.; n = 12–17 cells, analyses of variance (ANOVA); mean number of GAD puncta/40 μm dendrite: WT, 14.8 ± 1.3, GODZ KO, 14.8 ± 1.1; p, n.s.; n = 12–17, ANOVA). Similarly, inhibitory synapse formation was unaffected as indicated by normal colocalization of punctate postsynaptic γ2 staining and presynaptic GAD immunoreactivity (colocalization of γ2 with GAD in WT 92.0 ± 3.1% versus GODZ KO 90.0 ± 4.2%; p, n.s.; n = 12–18, ANOVA) (Fig. 2, a–d versus e–h and o). The normal density of GABAARs clusters and inhibitory synapses in KO cultures at first appeared to conflict with our previous results obtained by shRNA- or dominant negative GODZ-mediated knockdown of GODZ that suggested a role for GODZ in clustering of GABAARs and inhibitory synapse formation (18.Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Lüscher B. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses.J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (138) Google Scholar). However, we recently showed that γ2-GABAARs are critically important for GABAergic synapse formation selectively under conditions where GABAAR-deficient neurons are forced to compete with WT neurons for presynaptic innervation by WT neurons (22.Ren Z. Sahir N. Murakami S. Luellen B.A. Earnheart J.C. Lal R. Kim J.Y. Song H. Luscher B. Defects in dendrite and spine maturation and synaptogenesis associated with an anxious-depressive-like phenotype of GABAA receptor-deficient mice.Neuropharmacology. 2015; 88: 171-179Crossref PubMed Scopus (29) Google Scholar), i.e. conditions that mimic cultures that were sparsely transfected with shRNA constructs. Thus, to assess whether GODZ was required for inhibitory synapse formation under competitive conditions, we co-cultured GODZ KO neurons with WT neurons (1:9 ratio). WT neurons were prepared from a GFP transgenic embryo to facilitate distinction of mutant and WT neurons. Interestingly, under these conditions, GODZ KO neurons showed drastically reduced density of puncta for the γ2 subunit and GAD compared with WT cultures (density of puncta in KO neurons mixed with WT neurons as a percentage of pure WT cultures: γ2, 57.5 ± 4.9%; GAD, 57.9 ± 4.0%; p < 0.001, n = 10–12 cells for both comparisons, ANOVA, Tukey tests) (for absolute values, see Fig. 3, m and n) (Fig. 3, a–d versus i–l, m, and n). Residual GABAAR clusters in mutant neurons of mixed cultures remained invariably colocalized with presynaptic GAD (percent colocalization: GODZ KO/WT co-cultures, 89.2 ± 4.3%; WT pure cultures, 91.9 ± 3.0%; p, n.s.; n = 12–18, ANOVA) (Fig. 3o). Notably, our analyses of mutant neurons focused on pyramidal shaped neurons that lacked somatic GAD immunoreactivity and GFP fluorescence. Therefore, the GABAergic terminals contacting mutant neurons in these mixed genotype cultures originated almost exclusively from WT neurons, and the synaptic phenotypes of mutant pyramidal cells can be attributed specifically to postsynaptic defects in palmitoylation. The data indicate that GODZ KO neurons replicate the phenotype previously described for GODZ shRNA- and GODZ dominant negative construct-transfected cultures. Unaltered clustering of the γ2 subunit in (pure) GODZ KO cultures was unexpected and appeared inconsistent with a simple role of palmitoylation in forward trafficking of GABAARs to the plasma membrane, raising the possibility of functional redundancy of GODZ with SERZ-β. To further assess whether the phenotype of GODZ KO neurons was compromised by functional redundancy between GODZ and SERZ-β, we repeated the above experiments with neuron cultures prepared from DKO (GODZ−/− SERZ-β−/−) embryos. Similar to results obtained with GODZ KO cultures, cortical cultures (18 DIV) prepared from pure DKO embryos showed normal density and colocalization of punctate immunoreactivity for the γ2 subunit and GAD (density as percentage of WT: γ2, 97.4 ± 3.1%; GAD, 96.2 ± 3.6%; p, n.s.; n = 29–46 cells; colocalization of γ2 and GAD: DKO, 87.2 ± 1.5%; WT, 87.3 ± 1.9%; n = 30–46; p, n.s.; ANOVA) (Fig. 3, a–e versus e–h and m–o). Moreover, DKO neurons co-cultured with GFP-tagged WT neurons showed defects in pre- and postsynaptic clustering comparable with GODZ KO/WT co-cultures described above (density of puncta in DKO/WT co-cultures as a percentage of pure WT cultures: γ2, 49.7 ± 1.9%, n = 30–59; GAD, 55.3 ± 2.2%, n = 29–59; p < 0.001, ANOVA, Tukey tests) (Fig. 3, e–h versus i–l, m, and n). However, unlike in the case of GODZ KO/WT co-cultures, neurons of DKO/WT co-cultures showed significantly reduced colocalization of punctate γ2 and GAD immunoreactivities (colocalization in DKO/WT co-cultures as a percentage of pure WT neurons, 82.6 ± 2.9; p < 0.001, n = 30–59) (Fig. 3o). The size of γ2 puncta in DKO neurons of DKO/WT co-cultures remained unaltered (DKO neurons co-cultured, 96.8 ± 5.6% of WT; p, n.s.; n = 30–59, ANOVA). The data suggest that the reduced density of puncta observed in GODZ KO and GODZ/SERZ-β DKO neurons that was observed selectively in co-cultures was not simply a consequence of reduced forward trafficking of GABAARs to the cell surface. Curiously, quantitative comparison of the γ2 clustering defect observed in co-cultured GODZ KO neurons (density of γ2 puncta in GODZ KO, 57.5% of WT) with the defect observed in co-cultured DKO neurons (density of γ2 puncta in DKO, 49.7% of WT) revealed no significant difference (p, n.s; n = 30–59 (DKO) and n = 12–18 (GODZ KO), two-way ANOVA) and indicated that SERZ-β did not contribute to p" @default.
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• W2550519436 title "Dissociation of Golgi-associated DHHC-type Zinc Finger Protein (GODZ)- and Sertoli Cell Gene with a Zinc Finger Domain-β (SERZ-β)-mediated Palmitoylation by Loss of Function Analyses in Knock-out Mice" @default.
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