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Sudden death due to paralysis and synaptic and behavioral deficits when Hip14/Zdhhc17 is deleted in adult… Sanders, Shaun S; Parsons, Matthew P; Mui, Katherine K N; Southwell, Amber L; Franciosi, Sonia; Cheung, Daphne; Waltl, Sabine; Raymond, Lynn A; Hayden, Michael R Dec 7, 2016

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RESEARCH ARTICLE Open AccessSudden death due to paralysis andsynaptic and behavioral deficits whenHip14/Zdhhc17 is deleted in adult miceShaun S. Sanders1, Matthew P. Parsons2,3, Katherine K. N. Mui1, Amber L. Southwell1, Sonia Franciosi1,Daphne Cheung1, Sabine Waltl1, Lynn A. Raymond2 and Michael R. Hayden1*AbstractBackground: Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is animportant regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have beenimplicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis,Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylatesmany synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype isdevelopmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 insynaptic function in the adult.Results: Ten weeks after Hip14 deletion (iHip14Δ/Δ), mice die suddenly from rapidly progressive paralysis. Prior todeath the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptomindicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitterrelease and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice alsohave increased brain weight due to microgliosis and astrogliosis in the cortex.Conclusions: Behavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14Δ/Δ mice,and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. Thesedata suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.Keywords: Huntington’s disease, Palmitoylation, Palmitoyl acyltransferase, HIP14, DHHC17BackgroundIn recent years palmitoylation has emerged as an im-portant regulator of protein localization and function,particularly in neurons [1, 2]. Palmitoylation is the re-versible addition of long chain fatty acids, typicallypalmitate, to proteins at cysteine residues [3, 4]. It ismediated by DHHC-domain containing palmitoyl acyl-transferases (PATs) that palmitoylate proteins at cysteineresidues via a thioester bond [5, 6]. Many PATs have beenimplicated in diseases of the nervous system, includingHuntington disease (HD), an autosomal dominant fatalneurodegenerative disease; schizophrenia; amyotrophiclateral sclerosis; Alzheimer disease; and X-linked intellec-tual disability [1, 2].Palmitoylation is the only reversible lipid modifica-tion, and this reversibility is analogous to phosphoryl-ation, where enzyme-mediated addition and removal ofpalmitate allows for rapid cycling of palmitate on someproteins, providing an additional level of regulation oflocalization and function [7]. Indeed, in neurons, palmi-toylation has been shown to regulate localization ofmany synaptic proteins. For example, palmitoylation ofpost-synaptic density protein 95 (PSD95) is required forits synaptic localization, and its palmitoylation undergoescycles of de/repalmitoylation that regulate PSD95 nanoclus-ters within the synapse [8]. Palmitoylation also regulatesthe synaptic insertion/removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)* Correspondence: mrh@cmmt.ubc.ca1Centre for Molecular Medicine and Therapeutics, Department of MedicalGenetics, Child & Family Research Institute, University of British Columbia(UBC), Vancouver, BC V5Z 4H4, CanadaFull list of author information is available at the end of the article© Sanders et al. 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Sanders et al. BMC Biology  (2016) 14:108 DOI 10.1186/s12915-016-0333-7subunits GluA1 and GluA2, and of N-methyl-D-aspartatereceptor (NMDAR) subunits GluN2A and GluN2B [9, 10].Huntingtin interacting protein 14 (HIP14 or ZDHHC17)is the most highly conserved of the 23 human PATs. Itpalmitoylates many synaptic proteins, including cyst-eine string protein (CSP), GluA1, GluA2, PSD95,synaptosomal-associated protein 25 (SNAP25), synap-totagmin 1 (SYT1), the large conductance calcium- andvoltage-activated potassium BK channel (KCNMA1)STREX isoform, and the HD disease-causing proteinhuntingtin (HTT) [2]. It has recently become more appar-ent that HIP14 is an important regulator of synaptic func-tion. Indeed, Hip14 knockdown reduces PSD95 clusteringin neurons [6] and in Drosophila melanogaster HIP14 isrequired for CSP targeting to synaptic vesicles and, inturn, pre-synaptic exocytosis [11]. Interestingly, in an HDmouse model HIP14 is less active [12, 13] and the consti-tutive Hip14-deficient mouse (Hip14gt/gt) has behavioral,neuropathological, and synaptic dysfunction reminiscentof HD [12, 14, 15].The Hip14gt/gt mouse is a hypomorph expressing ~10%of endogenous HIP14 protein [16, 17] and the phenotypeis developmental, as neurodegeneration occurs during lateembryogenesis. Thus, we sought to determine the conse-quences of complete loss of Hip14 in the adult animal andits effect on synaptic deficits and neuronal degeneration.An inducible Hip14-deficient mouse model was generated,and Hip14 deletion was induced in the young adultmouse.ResultsGeneration of post-development Hip14-deficient miceHip14 “conditional knockout” (Hip14F/F) mice (Fig. 1a–d)were crossed to ubiquitously expressed tamoxifen(TM)-inducible Cre recombinase (Cre-ERT2)-expressingtransgenic mice [18]. Hip14 deletion was induced inHip14F/F;Cre + mice at 6 weeks of age by TM treatment(iHip14Δ/Δ herein) to allow mice a month to recoverfrom TM toxicity prior to any behavior testing per-formed at 3 months of age [19].Hip14mRNA and protein levels were assessed at 10 daysafter the last injection and 6 weeks post-induction to as-sess deletion efficiency compared to Hip14F/F;Cre– TMcontrol mice (iHip14F/F herein). Hip14mRNA and proteinexpression was decreased by >90% 10 days post-TM treat-ment in all brain regions and peripheral tissues tested(Fig. 1e and f). Greater than 95% loss of HIP14 proteinwas observed in the whole brain at 6 weeks post-TMtreatment (Fig. 1f). These data indicate that deletion ofHip14 in iHip14Δ/Δ mice is >90% effective.Low body weight and hyperactivity in iHip14Δ/Δ miceTo assess overall health, iHip14Δ/Δ mice were weighed at3 months of age, approximately 7 weeks post-induction.Both female and male iHip14Δ/Δ mice were approximately10% smaller than wild-type (WT) vehicle (VEH)-treatedand iHip14F/F control mice (Fig. 2a and b). To assessglobal nervous system and motor function, spontaneousactivity was assessed during the dark phase. iHip14Δ/Δmice were hyperactive during exploration of a novel envir-onment (increased distance traveled, Fig. 2c, and ambula-tory time, d).Motor coordination and sensorimotor gating deficits iniHip14Δ/Δ miceTo determine if loss of HIP14 in the adult mouse resultsin neurological dysfunction, motor function was assessed.Motor coordination of iHip14Δ/Δ mice was tested onrotarod and climbing tests [20]. iHip14Δ/Δ mice had motorcoordination deficits on the rotarod compared to controlWT VEH and iHip14F/F mice (Fig. 3a). As the rotarod per-formance is a trained test where mice learn to stay on therotarod, it is less sensitive to motor dysfunction than thespontaneous test of motor coordination: climbing [21, 22].There was also a dramatic reduction in the number ofclimbing events in these mice (Fig. 3b) but no change inthe number of rearing events, indicative of motivation toexplore the apparatus (Fig. 3c). Taken together, these dataindicate motor dysfunction.Schizophrenia and other neurological disorders wererecently shown to be enriched for palmitoylated proteins[1]. Pre-pulse inhibition (PPI) is a test of sensorimotorgating, partly mediated by the striatum [20, 23]. PPI defi-cits are associated with schizophrenia and other psychi-atric disorders as well as HD [24]. When a quieter tone(the pre-pulse) is played prior to a loud stimulus (thestartle pulse), mice with intact sensorimotor gating willstartle less than they would to the loud startle stimulusalone [25]. iHip14Δ/Δ mice showed impaired pre-pulseinhibition at all pre-pulse levels that was significant at 2,4, and 9 dB above background with a trend at 16 dBcompared to control mice (Fig. 3d), indicating impairedsensorimotor gating and potential striatal dysfunction.Increased escape response and anhedonia in iHip14Δ/Δ miceAs it is becoming increasingly evident that palmitoylationis important in neuropsychiatric disorders [1, 2], the im-pact of loss of HIP14 on psychiatric phenotypes such asdepression and anxiety was assessed [21, 26]. iHip14Δ/Δmice were tested in the Porsolt forced swim test for de-pression [26–28]. Interestingly, iHip14Δ/Δ mice spent dra-matically less time immobile during forced swimmingthan controls (Fig. 4a). During behavior testing iHip14Δ/Δmice were observed to be very reactive to the experi-menter and testing conditions, having explosive responsesto both. Thus, rather than truly reflecting an anti-depressive effect, these data in the forced swim test areSanders et al. BMC Biology  (2016) 14:108 Page 2 of 13consistent with the hyperactivity and reactivity to testingobserved in these mice.Anxiety-like behavior was assessed in the open field ex-ploration test, a well-established test of anxiety-like behav-iors in rodents [21, 29]. iHip14Δ/Δ mice explored thebrightly lit open field to the same extent as control mice,as measured by distance traveled (Fig. 4b), but spent lesstime in the center of the field (Fig. 4c), suggesting an in-crease in anxiety-like behaviors in these mice.To confirm anxiety in iHip14Δ/Δ mice, the mice weretested using the elevated plus maze (EPM) test for anxiety[30, 31]. Surprisingly, the iHip14Δ/Δ mice spent more timein the open arms of the EPM than the control WT VEHor iHip14F/F mice (Fig. 4d), suggesting decreased anxiety,Fig. 1 Generation of Hip14 conditional knockout mice. The targeting vector that was used is shown in (a). It was generated using PCR cloning ofthe 5′ and 3′ homology arms (5.5 and 3.2 kbp, respectively) and the deletion region (conditional knockout region [cKO]) with the indicatedrestriction enzyme sites added by PCR and used for cloning into the targeting vector, such that loxP sites are oriented in the same direction upand downstream of the cKO region. The deletion region, in gray, includes exon 2 and upstream and downstream intronic sequences to a total of1.1 kbp. The targeting vector also includes a positive neomycin (Neo) selection cassette flanked by flippase recognition target (frt) sites and anegative diphtheria toxin A (DTA) selection cassette outside of the homology arms. The wild-type (WT) allele is shown in (b) with the 5′ and 3′homology arms and the cKO region indicated. The recombined allele (Hip14F) is shown in (c). The neo cassette was removed during embryoniccell culture after targeting by electroporation with Flp recombinase and negative selection with G418. The knockout allele following expression ofCre recombinase is shown in (d) where recombination between the loxP sites occurs by the action of Cre leading to deletion of the cKO region,including exon 2. This deletion results in a frameshift mutation and multiple premature stop codons. The expression of Hip14 mRNA in the striatum,hippocampus, cortex, cerebellum, spleen, liver, kidney, and heart in iHip14Δ/Δ mice and in iHip14F/F control mice relative to β-actin is shownin (e) (N = 3). There was a 90% or greater decrease in Hip14 mRNA in iHip14Δ/Δ mice compared to control mice. f HIP14 protein expressionin the striatum, hippocampus, cerebellum, and cortex from iHip14Δ/Δ and iHip14F/F mice, where the tissues were collected 10 days post-TMtreatment, is shown. Also shown is whole brain collected 6 weeks post-TM treatment from iHip14Δ/Δ and iHip14F/F mice. Whole cell lysatewas run on western blot and probed with anti-HIP14 and anti-β-tubulin antibodies. The HIP14 immunoblot is the top panel in each set ofimages; the β-tubulin is in the bottom panel. The amount of HIP14 protein expressed is quantified in the graph relative to β-tubulin expression(N = 2–10). A 90% or greater decrease in HIP14 was observed in iHip14Δ/Δ mice. Data were analyzed using Student’s t testSanders et al. BMC Biology  (2016) 14:108 Page 3 of 13opposite to the findings from the open field testing. TheiHip14Δ/Δ mice did not explore the EPM as much as thecontrol mice (Fig. 4e), likely because they spent more timedipping their head off the edge of the open arms of themaze (Fig. 4f), again suggesting decreased anxiety. Thesedata suggest an anxiolytic phenotype rather than theanxiogenic phenotype suggested by the open field explor-ation test. Alternatively, the iHip14Δ/Δ mice may be tryingto escape the testing apparatus; i.e., they spend more timeexploring the edges of the open field box trying to find away out, and in the EPM they dip their heads off the openarms trying to escape the maze. This interpretation wouldalso be consistent with their reactivity to handling andtesting and reduced time spent immobile in the forcedswim test.To separate anxiety-like behavior from increased escaperesponse, a modified light-dark box test was designed thatcompletely removed any possibility of escape, where oneside was dark and the other side was brightly lit, and bothsides were completely enclosed. The iHip14Δ/Δ mice spentthe same amount of time in the light box as the controlmice (Fig. 4g). These data suggest iHip14Δ/Δ mice are notanxious per se but have an increased escape response.To delineate escape response from depressive-like be-havior, the iHip14Δ/Δ mice were tested using the sucrosepreference test for anhedonia-like behavior (the inabilityto experience pleasure), as anhedonia is a major symptomof depression [26, 32]. The sucrose preference test isperformed in the home cage with no experimenterpresent, thus eliminating the confound of increased escaperesponse. The iHip14Δ/Δ mice consumed the sameamount of fluid (Fig. 4g) but had decreased preference forsucrose compared to the control mice (Fig. 4h), indicatinganhedonia and suggesting a depressive-like phenotype.Increased forebrain weight, increased cortical volume,and decreased corpus callosum volume in iHip14Δ/Δ miceTo determine the effect of loss of HIP14 in the adultmouse on brain morphology and neurodegeneration,neuropathological assessments were performed. Increasedbrain weight was observed in iHip14Δ/Δ mice (Fig. 5a).This increase was restricted to the forebrain (Fig. 5b), asthere was no change in cerebellar weight compared toFig. 3 Motor coordination and sensorimotor gating deficits in 3-monthold iHip14Δ/Δ mice. Over three consecutive days, 3-month-old mice weretrained on a fixed-speed rotarod and tested on an accelerating rotarodon the fourth day (a). The number of falls (left) and the latency to thefirst fall (right) were recorded. iHip14Δ/Δ mice fell off the rotarod more(two-way ANOVA: genotype p = 0.0053, p < 0.0001; interactionp= 0.9071; N= 14–16) and sooner (two-way ANOVA: genotype p=0.0009, training day p< 0.0001, interaction p = 0.4581; N= 14–16) bothduring training and during testing. Mice were allowed to freely exploreand climb a wire mesh container. iHip14Δ/Δ mice climbed fewer times(b; ANOVA: p = 0.001; N = 13–18) but did not rear less (c; ANOVA: p =0.65; N= 13–18) compared to control mice. Pre-pulse inhibition (PPI)was measured as the percentage of decrease in the magnitude(velocity) of startle with a pre-pulse compared to the magnitude ofstartle without a pre-pulse. iHip14Δ/Δ mice had a PPI deficit compared tocontrols (d; repeated measures ANOVA: genotype p < 0.0001, pre-pulsep< 0.0001; N= 15–18)Fig. 2 Decreased body weight and hyperactivity in 3-month oldiHip14Δ/Δ mice. A ~10% decrease in body weight of female (a;analysis of variance, ANOVA: p < 0.0001) and male (b; ANOVA:p < 0.0001) iHip14Δ/Δ mice compared to WT VEH and iHip14F/F micewas observed (N = 16–24). Spontaneous activity was assessed byinfrared beam breaks during a 30-min exploration of a 27 × 27 ×20.3 cm box at 3 months of age. Total distance traveled (c) andambulatory time as assessed by consecutive beam breaks (d) wereassessed. On both measures the iHip14Δ/Δ mice were hyperactivecompared to controls (distance traveled ANOVA: p = 0.0005;ambulatory time ANOVA: p=<0.0001; ambulatory episodes ANOVAp= 0.0001; N= 13–18)Sanders et al. BMC Biology  (2016) 14:108 Page 4 of 13WT VEH control mice (Fig. 5c). Also, unexpectedly,there was no change in striatal volume in the iHip14Δ/Δmice compared to controls (Fig. 5d), but there was anincrease in cortical volume (Fig. 5e) and a decreasedcorpus callosum volume, indicating loss of white matter(Fig. 5f ) potentially due to axonal degeneration or lossof myelination.To understand what factors may account for the ob-served increase in cortical volume, astrocytes andmicroglia were assessed by glial fibrillary acidic protein(GFAP) and ionized calcium-binding adapter molecule1 (IBA1) staining intensity, respectively. There was adramatic increase in both GFAP (Fig. 5g and i) andIBA1 (Fig. 5h and j) staining intensity in the cortex ofiHip14Δ/Δ mice compared to controls, indicating signifi-cant astrogliosis and microgliosis, respectively.Impaired synaptic transmission in the striatum ofiHip14Δ/Δ miceSince palmitoylation has been implicated in localizationof synaptic proteins and in synaptic signaling and HIP14was previously shown to be important for striatal physi-ology and striatal processing during motor behaviors[14, 15], the synaptic properties of medium-sized spinyneurons (MSNs) in the striatum of iHip14Δ/Δ mice wereexamined [14, 33–35] by making current- and voltage-clamp recordings in the dorsal striatum. We observedno significant effect of loss of Hip14 on either MSN rest-ing membrane potential or rheobase (Fig. 6a–c): theamount of current injection required to initiate actionpotential firing. Membrane capacitance, an indirectmeasure of cell-surface area, was also similar betweengroups (Fig. 6d). Thus, MSN membrane potential, excit-ability, and cell size appear to be unaltered by loss ofHip14 in adulthood.To assay excitatory synaptic function, AMPAR-mediatedspontaneous excitatory post-synaptic currents (sEPSCs)were recorded from MSNs held at –70 mV in the presenceof picrotoxin, a γ-aminobutyric acid A (GABAA) receptorantagonist. There was a significant decrease in the fre-quency and a significant increase in the amplitude ofsEPSCs recorded from iHip14Δ/Δ MSNs (Fig. 6e and f)compared to controls. These data demonstrate synapticdysfunction in iHip14Δ/Δ mice and suggest a reduction inthe number of excitatory synapses and/or a reduction intransmitter release probability with additional AMPARs atthe synapses or more glutamate released per synapticvesicle.To assess transmitter release probability from corticalafferents onto MSNs in the striatum, a stimulating elec-trode was placed 200–250 μm dorsal to the recordedcell, various inter-pulse intervals were applied, and thepaired pulse ratio (PPR) was calculated. MSNs fromiHip14Δ/Δ mice had increased PPRs compared to MSNsfrom control mice (Fig. 6g and h). These data are indi-cative of a lower probability of transmitter release andare consistent with the reduction in sEPSC frequency,further suggesting synaptic dysfunction.Reduced survival due to rapidly progressing paralysis iniHip14Δ/Δ miceAs mice were being aged for longitudinal behavior studies,a dramatic decrease in survival of iHip14Δ/Δ mice, begin-ning at about 16 weeks of age or 10 weeks post-Hip14 de-letion, was observed (Fig. 7a). Typically, all mice appearedhealthy prior to sudden death. Six mice were found withhind limb paralysis prior to being euthanized for otherFig. 4 Three-month-old iHip14Δ/Δ mice have increased escaperesponse and display anhedonic-like behavior. iHip14Δ/Δ mice spentsignificantly less time immobile in the forced swim test (a; ANOVA:p = 0.0272; N = 15–19). Mice were placed in an open field underbright lighting. iHip14Δ/Δ mice explored the field to the same extentas the control mice (b; ANOVA p = 0.0844; N = 24–25) but spent lesstime in the center (c; ANOVA: p < 0.0001; N = 24–25). iHip14Δ/Δ micespent more time in the open arms of the elevated plus maze (d;ANOVA: p < 0.0001; N = 25–34; EPM), explored the maze less (e; ANOVA:p = 0.0007; N = 25–34), and dipped their heads off the edge of theopen arms more (f; ANOVA: p = 0.0001; N = 25–34) than controls. Micewere placed in an enclosed box with a brightly lit side and a dark side.iHip14Δ/Δ mice spent the same amount of the time in the light box ascontrol mice (g; ANOVA: p = 0.58; N = 13–16). Mice were allowedfree access to a 2% sucrose solution and water over a 24-h period,and the total fluid consumption (g/kg of body weight; h) was mea-sured. iHip14Δ/Δ mice had no change in total fluid intake (h;ANOVA: p = 0.27; N = 14–16) but had decreased preference for thesucrose solution (i; ANOVA: p = 0.0066; N = 14–16)Sanders et al. BMC Biology  (2016) 14:108 Page 5 of 13purposes. Post-mortem examination of 14 iHip14Δ/Δ micerevealed signs of paralysis in 13, including splayed hindlimbs and clenched front paws. One iHip14Δ/Δ mousefound with hind limb paralysis was monitored by video(Additional file 1). The paralysis progressed rapidly over5 h beginning with the hind limbs. Initially, the mouse didnot appear distressed and was able to move around thecage and eat. Paralysis progressed until the mouse couldno longer move and it was euthanized. A second iHip14Δ/Δ mouse was found almost completely paralyzed and wasvideo monitored for a few minutes until it went into re-spiratory arrest and died (Additional file 2). These data in-dicate that iHip14Δ/Δ mice have dramatically reducedsurvival due to rapidly progressing paralysis and suddendeath.Two iHip14Δ/Δ mice survived past 20 weeks of age(10%). One of these mice reached a humane endpointdue to wasting at 43 weeks of age and was euthanized.At the time of euthanasia, it weighed 30% less than itscontrol littermates. The brain was harvested for bio-chemistry to assess HIP14 protein levels to ensurecomplete loss of HIP14. Indeed, negligible HIP14 proteinwas detected, indicating that efficient recombinationoccurred in this mouse (Fig. 7b).DiscussionThe most striking phenotype of the iHip14Δ/Δ mice isthe rapidly progressing hind limb paralysis leading tosudden death. This was highly unexpected, as there is nosurvival deficit of Hip14gt/gt mice. HIP14 is the mosthighly conserved PAT, with 99% protein sequence iden-tity between human and mouse and 88% betweenhuman and zebrafish as well as 100% conservation ofthe DHHC active site domain from human to chicken[2]. This high sequence conservation suggests an essen-tial function for the protein, which is supported by thephenotype of iHip14Δ/Δ mice. The constitutive Hip14-deficient Hip14gt/gt mouse has HD-like neurological defi-cits [12, 20, 25]; thus, iHip14Δ/Δ mice were expected todevelop a similar phenotype. However, the severe pheno-type of iHip14Δ/Δ mice shows that HIP14 is crucial forthe life of the adult mouse. Hip14gt/gt mice develop earlyonset neurological disease, and these mice express 10%of the endogenous levels of HIP14 in all cells [16, 17],whereas complete loss in >90% of cells in iHip14Δ/Δadult mice causes a severe phenotype, including suddendeath. Thus, complete loss of HIP14 is likely not com-patible with survival. It will be interesting to see whathappens if HIP14 is fully deleted from conception. How-ever, there may also be developmental compensationthat occurs when HIP14 is deleted from conception,likely by other PATs, which cannot occur when HIP14 isdeleted in the adult animal.The iHip14Δ/Δ mice have motor coordination deficitssimilar to those of Hip14gt/gt mice. The motor deficitsare dramatic, particularly in the spontaneous climbingFig. 5 Three-month-old iHip14Δ/Δ mice have increased brain and forebrain weight, increased cortical volume, and decreased corpus callosumvolume. iHip14Δ/Δ mice have increased brain weight compared to control mice (a; ANOVA: p = 0.0004; N = 13–18), larger forebrain weight (b;ANOVA: p = 0.0002; N = 13–18), and unchanged cerebellum weight (c; ANOVA: p < 0.0001; N = 12–18). Brains were then sectioned and stainedwith NeuN to stain neurons, and striatal (d), cortical (e), and corpus callosum (f; CC) volume were determined. No change in striatal volume wasobserved (d; ANOVA: p = 0.43; N = 18–23) in iHip14Δ/Δ mice, but there was a significant increase in cortical volume (e; ANOVA: p < 0.0001; N = 13–18) and decrease in corpus callosum volume (f; ANOVA: p < 0.0001; N = 13–18). Sections were stained with antibodies against glial fibrillary acidicprotein (GFAP) or ionized calcium-binding adapter molecule 1 (IBA1) to stain astrocytes (g and i) and microglia (h and j). There was increasedstaining intensity of GFAP (g and i; ANOVA: p < 0.0001; N = 7–9) and IBA1 in iHip14Δ/Δ cortex (h and j; ANOVA: p = 0.0001; N = 7–9)Sanders et al. BMC Biology  (2016) 14:108 Page 6 of 13test. Also, similar to Hip14gt/gt mice [12, 25], iHip14Δ/Δmice have sensorimotor gating deficits. The PPI test as-sesses the ability to inhibit an unwanted motor responseto a stimulus and is believed to be mediated by the stri-atum [23]. Both motor dysfunction and PPI impairmentsuggest striatal dysfunction in the iHip14Δ/Δ mice.The psychiatric phenotype of the iHip14Δ/Δ mice alsosuggests striatal dysfunction. The iHip14Δ/Δ mice arehyperactive and reactive to handling, which is consistentwith the increased escape response observed during testsof anxiety and depression. This was confirmed wheniHip14Δ/Δ mice performed similarly to control mice in themodified light-dark box test that eliminated any avenuesfor escape. However, anxiety and escape response arelikely associated. Thus, it is possible that they becomeanxious when they are unable to escape a novel environ-ment [36]. Overall, the increased escape response pheno-type of iHip14Δ/Δ mice agrees with rodent striatal lesionmodels with enhanced escape response behavior, provid-ing further evidence of an essential role for HIP14 in stri-atal function [37].Interestingly, iHip14Δ/Δ mice have increased forebrainweight due to microgliosis and astrogliosis in the cortex.This may be a downstream response to neuron or circuitdysfunction or may suggest a novel role for HIP14 inglial cell function. Also, although there is clear striataldysfunction in iHip14Δ/Δ mice, there was no change instriatal volume, unlike the striatal atrophy observed inHip14gt/gt mice [12, 38]. One possible explanation forthis discrepancy is that iHip14Δ/Δ mice die before suffi-cient striatal neuron death for detection by stereologyhas occurred. There was, however, a decrease in corpuscallosum volume, suggesting decreased white matter andpotentially axonal degeneration or demyelination.Further evidence for striatal dysfunction was apparentin the physiology of MSNs. Although there is no changein membrane excitability or surface area in iHip14Δ/ΔMSNs, they did display aberrant synaptic transmission.The increase in sEPSC amplitude, decrease in sEPSC fre-quency, and increased PPR suggest a lower probabilityFig. 7 Reduced survival in iHip14Δ/Δ mice and HIP14 proteinexpression in the brain of a 43-week-old iHip14Δ/Δ mouse. A dramaticreduction in survival was observed in the iHip14Δ/Δ mice compared tocontrols (a; log-rank test: X2(4) = 93.76; N = 11–24). b Whole cell lysatewas run on western blot and probed with anti-HIP14 and anti-β-tubulin antibodies. The HIP14 immunoblot is the top panel and thatof the β-tubulin is in the bottom panel. The amount of HIP14 proteinexpressed is quantified in the graph relative to β-tubulin expression(N = 3 WT VEH, 2 iHip14F/F, and 1 iHip14Δ/Δ). A 97% decrease in HIP14was observed in the iHip14Δ/Δ mouse. The representative images arecomposites from the same western blot imageFig. 6 Impaired synaptic transmission in iHip14Δ/Δ MSNs.MSNs in thecentral dorsal striatum were whole-cell patch clamped in acute coronalslices from 3-month-old mice. A representative trace of current-clampmembrane potential responses to a series of current injections (from –100 pA to 200 pA in 50-pA increments) is shown in (a). iHip14Δ/Δ MSNshad the same resting membrane potential (RMP) (b: ANOVA: p = 0.68;N = 21–25), fired at the same rheobase current (c; ANOVA: p = 0.30; N= 21–25), and had the same membrane capacitance (d; ANOVA:p = 0.094; N = 31–43). iHip14Δ/Δ mice had decreased frequency(f left; ANOVA: p < 0.0001; N = 19–23) but increased amplitude (f right;ANOVA: p < 0.0001; N = 19–23) of spontaneous excitatory post-synapticcurrents (sEPSC), representative traces are shown in (e). iHip14Δ/Δ micehad increased paired pulse ratios (h; PPR; two-way ANOVA: genotypep = 0.0001; pulse interval p < 0.0001; interaction p = 0.0047; N = 8–10),representative traces are shown in (g)Sanders et al. BMC Biology  (2016) 14:108 Page 7 of 13of transmitter release but more AMPARs at excitatorysynapses in the striatum and/or more glutamate releasedper synaptic vesicle to the same number of AMPARs.Loss of palmitoylation at either palmitoylation site ofGluA1 or GluA2 AMPAR subunits would increase theirsynaptic expression, which could contribute to thesephenotypes [9].HIP14 has been shown to be a “hub” protein withmany interacting partners, and it shares many interac-tors (not specifically substrates) with HTT, also a “hub”protein with many interactors [39]. In addition to beinga PAT, HIP14 has also been shown to have other, non-PAT-related, functions in MAP kinase signaling andmagnesium/manganese transport [40–42]. Thus, thephenotype of these mice may be due to loss of palmitoy-lation of one or multiple crucial proteins, may resultfrom loss of one of these other functions of HIP14, ormay be caused by a combination of all these factors.ConclusionsThis is the first study, to our knowledge, to examine the“conditional knockout” of a DHHC PAT and conclusivelydemonstrates that HIP14 is essential for life and neuronalintegrity. The iHip14Δ/Δ mice have a severe phenotype,different than that of the Hip14gt/gt mice, that results insudden death, striatal dysfunction, and significant astro-gliosis and microgliosis. These data highlight the import-ance of this PAT to neurological function and suggest thatpalmitoylation is an essential protein modification.MethodsGeneration of inducible Hip14 knockout miceXenogen Biosciences (now Taconic Biosciences, Rensselaer,NY, USA) generated the Hip14 “floxed” mice (Hip14F)on the FVB/N background strain using a gene targetingstrategy where exon 2 was selected as the conditionaldeletion region, as deletion of this region leads to aframeshift mutation and multiple premature stop co-dons (Fig. 1a–d). The 5’ and 3’ homology arms and theconditional knockout region (cKO) were amplified frombacterial artificial chromosome DNA and inserted intothe targeting vector at the indicated restriction enzymesites such that the cKO region was flanked by loxP sites(Fig. 1a). A positive selection neo cassette was includedand flanked by flippase (Flp) recognition target (FRT)sites, and a negative selection cassette diphtheria toxinA (DTA) was also included to select against random in-sertion (Fig. 1a). Male FVB/N embryonic stem cellswere electroporated with the targeting vector and selectedusing G418 (Geneticin) resistance and screened for hom-ologous recombination at the 5′ and 3′ homology armswith the WT allele (Fig. 1b) by restriction enzyme digest,southern blot, and PCR. The neo cassette was thenremoved in positive clones by electroporation with Flprecombinase to mediate recombination between the FRTsites and generate the recombined Hip14F allele (Fig. 1c).Neo cassette deletion was confirmed by G418 sensitivityand PCR. Hip14F embryonic stem cells were then injectedinto C57BL/6 J blastocysts to generate male chimeras thatwere bred with FVB/N females. Resulting white coat pro-geny indicated germline transmission, and those mice weregenotyped using the following primers: the forward primerin the 5′ homology arm in intron 1 (5′-GGAGAATGGTTAGGAAAAGCTCGTACC-3′) and the reverse primer inthe cKO region in intron 1 upstream of the first loxP site(5′-GAGGAAAGCATGCAAGAGCACTTCTC-3′).Hip14F/F mice were then crossed to mice expressingCre-ERT2 under the human ubiquitin ligase C promoter, apromoter that will result in ubiquitous Cre expression inall cell types [18] (The Jackson Laboratory, Bar Harbor,ME, USA). The Cre-ERT2 transgene expresses Cre recom-binase fused to a mutated form of the estrogen receptorthat is not activated by estrogen but is activated by theestrogen analog tamoxifen (TM) [18]. This generatedmice in which Hip14 can be deleted at any time point(Hip14F/F;Cre-ERT2). The primers used to genotype atthe Cre-ERT2 transgene were 5′-GCGGTCTGGCAGTAAAAACTATC-3′ and 5′- GTGAAACAGCATTGCTGTCACTT-3′. Gene deletion was induced using a 5-day TM treatment paradigm by giving a single intraper-itoneal injection once a day for 5 days at a dose of0.2 mg TM/g body weight in 98% corn oil with 2%ethanol (iHip14F/F and iHip14Δ/Δ) or vehicle alone (WTVEH) as previously described [18]. Mice were treatedwith TM at 6 weeks of age.Quantitative real-time PCRTotal RNA was isolated from –80 °C frozen tissues usingthe RNeasy mini kit (Qiagen, Venlo, The Netherlands).RNA was treated with DNAse I (Life Technologies, Carls-bad, CA, USA) to remove residual genomic DNA. cDNAwas prepared from 1 μg total RNA using the SuperScript®III First-Strand Synthesis System (Life Technologies,Carlsbad, CA, USA). Quantitative RT-PCR (qPCR) on themouse Hip14 gene using primers spanning exons 1 and 2(5′-ACCCGGAGGAAATCAAACCACAGA-3′ and 5′-TACATCGTAACCCGCTTCCACCAA-3′) was performedusing Power SYBR Green PCR Master Mix (AppliedBiosystems, Life Technologies, Carlsbad, CA, USA) andthe ABI 7500 Fast Real-Time PCR System (Applied Bio-systems, Life Technologies, Carlsbad, CA, USA) underdefault conditions. Each sample was run in triplicate.Expression levels for mRNA were normalized to β-actin.AntibodiesThe primary antibodies used were HIP14 polyclonalantibody (in house, 1:400 for immunoblotting), β-tubulinmonoclonal antibody (T8328, Sigma, RRID:AB_1844090,Sanders et al. BMC Biology  (2016) 14:108 Page 8 of 131:5000 for immunoblotting), and NeuN antibody(MAB377, Millipore, RRID:AB_2298772, 1:1000). Biotinyl-ated anti-mouse antibody (BA-9200, RRID:AB_2336171,Vector Laboratories, 1:1000 for immunohistochemistry)was used as a secondary antibody for immunohisto-chemistry. Fluorescently conjugated secondary anti-bodies used for immunoblotting were Alexa Fluor 680 goatanti-rabbit (A21076, Molecular Probes, RRID:AB_2535736,1:10000) and IRDye 800CW goat anti-mouse (610-131-121,Rockland, RRID:AB_220123, 1: 2500).Tissue lysis and western blotting analysisTissues were homogenized on ice for 5 min in one volume1% SDS TEEN (TEEN: 50 mM Tris pH 7.5, 1 mM EDTA,1 mM EGTA, 150 mM NaCl, and 1× complete proteaseinhibitor cocktail [Roche]) and subsequently diluted infour volumes 1% TritonX-100 TEEN for 5 min for furtherhomogenization. Samples were sonicated once at 20%power for 5 s to shear DNA, and the insoluble materialwas removed by centrifugation at 14,000 rpm for 15 min.Proteins in cell lysates were heated at 70 °C in 1×NuPAGE LDS sample buffer (Invitrogen) with 10 mMdithiothreitol (DTT) before separation by SDS-PAGE.After transfer of the proteins onto a nitrocellulose mem-brane, immunoblots were blocked in 5% milk TBS (TBS:50 mM Tris pH 7.5, 150 mM NaCl). Primary antibodydilutions of HIP14 polyclonal antibody and β-tubulinmonoclonal antibody in 5% BSA PBST (bovine serum al-bumin, phosphate buffered saline with 5% Tween-20)were applied to the immunoblots at 4 °C overnight. Cor-responding secondary antibodies were applied in 5%BSA PBST for an hour. Fluorescence was scanned andquantified with an Odyssey Infrared Imaging system (Li-COR Biosciences, Lincoln, NE, USA) and quantified usingthe Li-COR software. All error bars represent standarderror of the mean.BehaviorAll behavioral testing was performed with the tester blindto genotype. All testing was performed at 3 months of age(7 weeks post-TM injection). All of the apparatuses werecleaned between mice with 70% ethanol.Spontaneous activitySpontaneous activity in the dark was measured usingthe Med Associates activity monitoring system (MedAssociates Inc., St Albans, VT, USA) as previously de-scribed [20]. Briefly, no later than 1 h after the begin-ning of the dark cycle following 1 h of acclimatizationto the room, mice were placed in the center of the test-ing chamber (27 × 27 × 20.3 cm) and allowed to freelymove about and explore for half an hour. A number ofautomated readouts were recorded. Ambulatory time isthe total time the mouse spent moving while makingconsecutive beam breaks and ambulatory episodes arethe number of times the mouse began ambulating froma resting position.Rotarod and climbingFixed-speed and accelerating rotarods were used to assessmotor coordination (Ugo Basile, Comerio, Italy) as previ-ously described [20]. Briefly, mice were trained once a dayfor 3 days on the fixed-speed 18-rpm rotarod for 120 s,and the number of falls and latency to the first fall wererecorded. On the fourth day, mice were tested on an ac-celerating rotarod that accelerates from 5 to 40 rpm over300 s, and the latency to fall was recorded. The average ofthree trials is reported.Motor coordination was also tested on the climbingapparatus as previously described [21]. Briefly, micewere placed inside a closed-top wire mesh cylinder(10 × 15 cm) on the tabletop and were allowed to freelyexplore for 300 s while being video recorded. A climb-ing event was recorded when all four paws were off thesurface of the tabletop and a rearing event was re-corded when the forepaws were off the surface of thetabletop. The climbing time was recorded as the totaltime from when the fourth paw left the tabletop towhen the first paw touched back down.Pre-pulse inhibition (PPI)PPI was performed using the Startle Response System(San Diego Instruments, San Diego, CA, USA) as previ-ously described [25]. Briefly, mice were placed in a star-tle chamber and allowed to acclimatize with backgroundnoise for 5 min. Mice were then exposed to six trials ofa 40-ms, 120-dB startle pulse to test the acoustic startleresponse. Mice were then exposed to eight blocks of six(48 trials in total) pseudorandomized trials of (1) nostimulus, (2) the 40-ms, 120-dB startle pulse alone, or(3–6) the 40-ms, 120-dB startle pulse preceded 100 msby a 20-ms pre-pulse of 2, 4, 8, or 16 dB above back-ground. An extra 40-ms, 120-dB pulse was given in fourof the eight blocks. Finally, the mice were exposed to an-other six trials of the 40-ms, 120-dB startle pulse. Theinter-trial interval was between 8 and 23 s and was pseu-dorandomized between trials. PPI is the percentage ofdecrease in the startle response when a pre-pulse isgiven prior to the startle pulse and was calculated as theaverage of six trials per pre-pulse as follows: PPI = [(startlepulse-alone startle) - (pre-pulse + startle pulse startle)]/pulse-alone startle.Porsolt forced swim testThe Porsolt forced swim test was used to assessdepressive-like behavior and was performed as previ-ously described [26–28, 43]. Briefly, mice were placed inindividual cylinders (25 cm tall × 19 cm wide) filled withSanders et al. BMC Biology  (2016) 14:108 Page 9 of 13room temperature water to a depth of 15 cm andallowed to swim for 6 min while being recorded by videocamera. The time spent immobile (not swimming) dur-ing the final 5 min was scored.Open fieldOpen field exploration was used as a test of anxiety-likebehaviors as previously described [21]. Briefly, mice wereplaced into the lower left corner of a 50 × 50 cm open grayPlexiglas box with 16-cm sides in a brightly lit room. Themice were allowed to explore the box for 10 min while be-ing recorded via ceiling-mounted video camera. Videoswere live scored using Ethovision XT 7 animal trackingsoftware (Noldus Information Technology), and the totaldistance traveled and the total time spent in the center ofthe field were scored as measures of exploratory activityand anxiety-like behavior, respectively.Elevated plus mazeEPM exploration was used as a test of anxiety-like behav-ior as previously described [31]. Briefly, mice wereplaced in the center of an EPM 50 cm off the groundwith 30 × 10 cm arms, two of which are enclosed by 20-cm walls. Mice were allowed to freely explore the mazefor 5 min while a ceiling-mounted camera recordedtheir activity and Ethovision XT 7 live scored the videos.Distance traveled was used to assess exploratory activity.The time spent in the open arms (open arm duration) andhead dips off the edges of the open arms were used asmeasures of anxiety-like behavior.Light-dark boxThe light-dark box was used to test for anxiety in anenvironment where escape was not possible, i.e., a com-pletely enclosed environment. The Gemini AvoidanceSystem (San Diego Instruments) was used for this pur-pose; no cues or shocks were used. The door betweenthe two chambers was kept open so mice could freelyexplore both sides of the box, and on one side a lightwas shone through the transparent door to create abrightly lit light box. The door on the other side wasblacked out to create a dark box. Mice were allowed tofreely explore the apparatus, and their activity was re-corded using a video camera through the light box side.The total time spent in the light box was scored as ameasure of anxiety-like behavior.Sucrose preferenceSucrose preference was used to test for anhedonia, orthe loss of pleasure-seeking behaviors, a symptom of de-pression, as previously described [26, 44]. Briefly, micewere single housed in a full-size cage and were given adlibitum access to food and to two water bottles. Micewere allowed to acclimatize to the bottles for 1 week.On day 7 the water in one of the bottles was replaced witha 2% sucrose solution and the mice and both bottles wereweighed. Twenty-four hours later the bottles wereweighed again and the total fluid and sucrose intake werecalculated as g/kg of body weight. Sucrose preference wascalculated as follows: sucrose preference = (sucrose intake/total fluid intake) × 100.NeuropathologyAll neuropathological studies were conducted as previ-ously described [12, 31]. Mice were anesthetized by in-traperitoneal injection of 2.5% avertin and intracardiallyperfused with ice-cold 4% paraformaldehyde. Brainswere harvested and post-fixed in 4% paraformaldehydefor 24 h at 4 °C, and then cryopreserved in 30% sucrosein phosphate-buffered saline (PBS). To determine thebrain weight, the olfactory bulbs, paraflocculi, and brainstem were removed prior to weighing. The cerebellumwas then removed and weighed separately. Forebrainweight was calculated as brain weight minus cerebellumweight. The forebrain was then flash frozen on dry ice,mounted with Tissue-TEK O.C.T. compound (SakuraFinetek, Torrance, CA, USA), and sectioned coronallyon a cryostat (Microm HM 500 M) into 25-μm free-floating sections. Sections were stored until immunohisto-chemical processing in PBS with 0.01% sodium azide.A series of 25-μm sections spaced 200 μm apart span-ning the striatum were processed for stereological volumet-ric assessments by staining with NeuN antibody (1:1000,Millipore MAB377) overnight at room temperature tostain all neuronal nuclei. Sections were then stained withbiotinylated anti-mouse antibody for 2 h (1:1000, VectorLaboratories BA-9200) and the signal was amplified usingthe Vectastain ABC kit for 30 min (1:1000, Vector Labora-tories) and then detected with 3,3′-diaminobenzidine(DAB, Thermo Scientific). StereoInvestigator software(Microbrightfield Bioscience, Williston, VT, USA) was usedto determine striatal, cortical, and corpus callosum vol-umes by tracing the perimeter of the desired structures;the volumes were determined using the Cavalieri principle.Two additional series of sections described above wereused for glial fibrillary acidic protein (GFAP) and ionizedcalcium-binding adapter molecule 1 (IBA1) immunohis-tology. Sections were blocked with 3% H2O2 in PBS for30 min, then washed with PBS. Sections were thenblocked with 5% normal goat serum (NGS) in PBS-Tritonfor 30 min. Sections were incubated overnight at roomtemperature in either monoclonal mouse anti-GFAP-Cy3antibody (1:500, Sigma-Aldrich) or polyclonal rabbit anti-IBA1 antibody (1:500, Wako), both solutions made in 1%NGS and PBS-Triton. Sections were then incubated for2 h at room temperature in either biotinylated goat anti-mouse IgG antibody (1:500, Vector) or biotinylated goatanti-rabbit IgG antibody (1:500, Vector), both solutionsSanders et al. BMC Biology  (2016) 14:108 Page 10 of 13made in 1% NGS and PBS-Triton. Lastly, sections wereincubated with the Vectastain ABC kit (1:1000, Vector)for 30 min and developed with DAB (1:10, Sigma-Aldrich)for 2 min. Sections were mounted on Superfrost Plusmicroscope slides (Fisher) prior to analysis. Sections wereimaged on a Zeiss Axioplan 2 imaging system with a 5×Zeiss Plan-Neofluar objective using a Photometrics CoolSnap HQ camera. Analyses were done using MetaMorphsoftware version 6.3 (Universal Imaging Corporation,Bedford Hills, NY). After delineating the cortex for eachimage, labeling of GFAP and IBA1 was identified usingthe threshold held at a constant level with background ex-cluded for all images and then analyzed using the “inte-grated morphometry” feature. Relative levels of GFAP andIBA1 staining were calculated as the sum of the integratedoptical density (IOD) for each image divided by the areaof the region selected, then multiplied by the samplinginterval (8) and section thickness (25 μm). No stainingwas observed in a negative control without primary anti-body [31].ElectrophysiologyMice were transferred to the University of BritishColumbia (UBC) Animal Research Unit approximately4–5 weeks following TM injections and all electro-physiological experiments were performed on mice thatwere approximately 3 months old. Electrophysiologicalanalyses were performed as previously described [14].Briefly, mice were anesthetized with isoflurane andbrains were quickly removed and immediately placed inan ice-cold cutting solution that contained (in milli-moles): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4,2.5 MgCl2, 0.5 CaCl2, and 10 glucose. Coronal blockscontaining the striatum were then cut on a vibratome(Leica VT1200S) at 400 μm. Striatal sections were trans-ferred to artificial cerebrospinal fluid (ACSF), which wasthe same as the cutting solution except that it contained1 mM MgCl2 and 2 mM CaCl2, and were heated toapproximately 32 °C for 30–45 min.Following recovery, slices were transferred to a record-ing chamber with ACSF perfused at a rate of 2–3 ml/min.In striatal sections, spiny projection neurons in the dorsalstriatum were targeted for recording [14]. For excitatorypost-synaptic current (EPSC) recordings in the striatum,picrotoxin (50 μM) was added to the ACSF, but tetrodo-toxin (TTX, 0.5 mM) was omitted, as we have previouslyshown that most EPSCs in our coronal slice preparationare action potential-independent [45]. However, whilelargely action potential-independent, these are referred toas spontaneous EPSCs (sEPSCs) in the manuscript to indi-cate the lack of TTX. Glass pipettes (3–6 MΩ) were filledwith a potassium gluconate (KGlu) internal solution forsEPSC recordings [46]. The liquid junction potential (the-oretical = –15.6 mV) was left uncorrected. sEPSCs werefiltered at 1 kHz and digitized at 20 kHz. Where applic-able, glutamate release was evoked by an ACSF-filledglass pipette (1 MΩ) placed 200–250 μm dorsal to therecorded cell. Paired pulse ratios (PPRs) were obtainedat a –70 mV holding voltage, and various inter-pulseintervals were applied with a stimulus intensity knownto generate a response approximately 30–40% of themaximal response. Basic membrane properties were ob-tained within 60 s following break-in by monitoring thecurrent response to a 10-mV voltage step applied in themembrane test feature in Clampex 10 (Molecular Devices,Sunnyvale, CA, USA). All electrophysiological recordingswere acquired and analyzed using the pClamp 10 softwarebundle.StatisticsData were analyzed using the Student’s t test, one-wayANOVA, or two-way ANOVA as indicated using Prism 5software where all post hoc tests in ANOVA analyses usedBonferroni’s multiple comparison test. Error bars indicatestandard error of the mean and in all graphs the mean isindicated. * p < 0.05, ** p < 0.01, *** p < 0.001. In all cases,except for Fig. 6, the number of replicates (N) refers to thenumber of individual mice used and is considered to meanbiological replicates. In Fig. 6, N refers to the number ofcells analyzed from a total of 4 mice per genotype.ANOVA values and exact numbers are listed in Additionalfile 3.Additional filesAdditional file 1: Rapidly progressing paralysis in an iHip14Δ/Δ mouseover the course of 5 h. An iHip14Δ/Δ mouse found with hind limbparalysis was monitored by video. The paralysis progressed rapidly over5 h, beginning with the hind limbs, from where the mouse was able tomove around the cage and eat until it could no longer move and waseuthanized. The video lasts a total of 7 min, with the first minute fromthe beginning of the 5 h and the rest of the video just before the mousewas euthanized. (MOV 12680 kb)Additional file 2: Sudden death due to rapidly progressive paralysis inan iHip14Δ/Δ mouse. A second iHip14Δ/Δ mouse was found almostcompletely paralyzed and was video monitored for a few minutes until itwent into respiratory arrest and died. (MOV 6931 kb)Additional file 3: One-way and two-way ANOVA values and replicates.(XLSX 40 kb)Additional file 4 Individual data values for experiments where N < 6.(XLSX 44 kb)Abbreviations(DNAJC5): DnaJ heat shock protein 40 homolog; ACSF: Artificial cerebrospinalfluid; AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidreceptor; ANOVA: Analysis of variance; cKO: Conditional knockout region ofHip14; CSP: Cysteine string protein; DARPP-32: Dopamine- and cAMP-regulated neuronal phosphoprotein (PPP1R1B); DHHC: Asp-His-His-Cys;DTA: Diphtheria toxin A; EPM: Elevated plus maze; EPSC: Excitatory post-synaptic current; Flp: Flippase recombinase; FRT: Flippase recognition target;FVB/N: FVB/NJ mouse strain, Friend virus B NIH Jackson; G418: Geneticin;GABAA: γ-aminobutyric acid A (receptor); GluA1: AMPA receptor subunit 1(GRIA1); GluA2: AMPA receptor subunit 2 (GRIA2); GluN2A: NMDA receptorSanders et al. BMC Biology  (2016) 14:108 Page 11 of 13subunit 2A (GRIN2A); GluN2B: NMDA receptor subunit 2B (GRIN2B);HD: Huntington disease; HIP14: Huntingtin interacting protein 14 (ZDHHC17);HTT: HD disease-causing protein huntingtin; loxP: Locus of X-over P1, 34 bpCre recombinase sequence from P1 bacteriophage; MAP: Mitogen-activatedprotein; MSN: Medium spiny neurons; NMDAR: N-methyl-D-aspartatereceptor; PAT: Palmitoyl acyltransferase; PPI: Pre-pulse inhibition; PPR: Pairedpulse ratio; PSD95: Post-synaptic density protein 95 (DLG4);SNAP25: Synaptosomal-associated protein 25; STREX BK: Stress-regulatedexon splice variant of the calcium- and voltage-activated potassium channel;SYT1: Synaptotagmin 1; TM: Tamoxifen; WT: Wild type.AcknowledgementsWe thank Mark Wang (Center for Molecular Medicine and Therapeutics[CMMT]) for assistance with animal care and husbandry, Weining Zhang(CMMT) for assistance with behavior testing, Nikola Lazic for technicalassistance, and Dr. Dale Martin (CMMT) for editing the manuscript.FundingThis work was supported by the CHDI Foundation, Inc. and the CanadianInstitutes for Health Research (CIHR grant number GPG-102165 to MRHand LAR). MRH is a University Killam Professor and a Canada ResearchChair in Human Genetics and Molecular Medicine. The authors declareno competing interests.Availability of data and materialsThe datasets generated and/or analysed during the current study areavailable from the corresponding author on reasonable request. Individualdata values in those experiments with N < 6 are given in Additional file 4.Authors’ contributionsSSS conceived of the project, analyzed and interpreted all biochemical andneuropathological data; produced, analyzed, and interpreted all behavior andsurvival data; assisted with the analysis and interpretation of theelectrophysiological data; prepared all of the figures; and wrote themanuscript. MPP produced, analyzed, and interpreted allelectrophysiological data and wrote the corresponding Methods sections.KKNM produced all biochemical data. ALS assisted with design andinterpretation of the behavior testing experiments and resulting data. SF,DC, and SW generated all neuropathological data and wrote thecorresponding Methods sections. LAR assisted with the interpretation ofthe electrophysiological data. MRH assisted with the conception of theproject, the experimental design, interpretation of the results, and thewriting of the manuscript. All authors read and approved the finalmanuscript.Competing interestsThe authors declare that they have no competing interests.Ethics approvalAll procedures and animal work were approved by the University of BritishColumbia Committee on Animal Care in protocols A12-0063 and A16-0130.Author details1Centre for Molecular Medicine and Therapeutics, Department of MedicalGenetics, Child & Family Research Institute, University of British Columbia(UBC), Vancouver, BC V5Z 4H4, Canada. 2Department of Psychiatry, BrainResearch Centre and Djavad Mowafaghian Centre for Brain Health, UBC,Vancouver, BC V6T 1Z3, Canada. 3Present address: Division of BiomedicalSciences, Faculty of Medicine, Memorial University, Newfoundland andLabrador A1B 3V6, Canada.Received: 12 September 2016 Accepted: 16 November 2016References1. Sanders SS, Martin DDO, Butland SL, Lavallée-Adam M, Calzolari D, Kay C,Yates JR, Hayden MR. 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J Biol Chem. 2014;289:3518–28.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Sanders et al. BMC Biology  (2016) 14:108 Page 13 of 13


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