Neurogenetics (2015) 16:277–285 DOI 10.1007/s10048-015-0450-4
ORIGINAL ARTICLE
Deletion of Inpp5a causes ataxia and cerebellar degeneration in mice Andy W. Yang 1 & Andrew J. Sachs 1 & Arne M. Nystuen 1
Received: 30 March 2015 / Accepted: 5 May 2015 / Published online: 9 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The progressive and permanent loss of cerebellar Purkinje cells (PC) is a hallmark of many inherited ataxias. Mutations in several genes involved in the regulation of Ca2+ release from intracellular stores by the second messenger IP3 have been associated with PC dysfunction or death. While much is known about the defects in production and response to IP3, less is known about the defects in breakdown of the IP3 second messenger. A mutation in Inpp4a of the pathway is associated with a severe, early-onset PC degeneration in the mouse model weeble. The step preceding the removal of the 4-phosphate is the removal of the 5-phosphate by Inpp5a. Gene expression analysis was performed on an Inpp5aGt(OST50073)Lex mouse generated by gene trap insertion using quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blot. Phenotypic analyses were performed using rotarod, β-galactosidase staining, and phosphatase activity assay. Statistical significance was calculated. The deletion of Inpp5a causes an early-onset yet slowly progressive PC degeneration and ataxia. Homozygous mutants (90 %) exhibit perinatal lethality; surviving homozygotes show locomotor instability at P16. A consistent pattern of PC loss in the cerebellum is initially detectable by weaning and widespread by P60. Phosphatase activity toward
Electronic supplementary material The online version of this article (doi:10.1007/s10048-015-0450-4) contains supplementary material, which is available to authorized users. * Andy W. Yang [email protected] Andrew J. Sachs [email protected] Arne M. Nystuen [email protected] 1
The Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA
phosphoinositol substrates is reduced in the mutant relative to littermates. The ataxic phenotype and characteristics neurodegeneration of the Inpp5aGt(OST50073)Lex mouse indicate a crucial role for Inpp5a in PC survival. The identification of the molecular basis of the selective PC survival will be important in defining a neuroprotective gene applicable to establishing a disease mechanism. Keywords Inpp5a . Knockout mouse . Cerebellar degeneration . Ataxia
Introduction In neurons, the central mechanism controlling calcium release from intracellular stores involves the generation of a second messenger, inositol 1,4,5-trisphosphate (IP3), in response to an extracellular signal [1]. There are multiple steps in this pathway, which are performed by proteins that are encoded by large gene families [2]. In cerebellar Purkinje cells, an excitatory glutamate signal from climbing fibers is transduced through metabotropic glutamate receptor 1 (mGluR1) [3, 4]. This causes the generation of IP3 via guanine nucleotidebinding protein, alpha q polypeptide (GαQ) [5], and phospholipase C beta 4 (PLCβ4) [6]. IP3 binds inositol 1,4,5-trisphosphate receptor 1 (ITPR1) and causes the release of Ca2+ (Fig. 1a) [7]. The above genes, involved in the generation and response to IP3, have been associated with neurological disorders in human and mouse [8, 9]; however, relatively less is known about perturbations in the breakdown of the IP3 signal. One such model, Inpp4awbl, is a recessive mouse mutant characterized by severe ataxia and pre-wean lethality. The Inpp4awbl mutant has a unique combination of neuroanatomical abnormalities including neurodegeneration in the cerebellum and hippocampus. Specifically, Purkinje cells are lost in
278
Neurogenetics (2015) 16:277–285
Fig. 1 Inpp5a is expressed in the cerebellar Purkinje cells and muscle. The pathway resulting in the release of Ca2+ from intracellular stores is shown in panel a, with the members of the pathway expressed in Purkinje cells. Phosphate groups (red dots) are removed from the inositol backbone by enzymes including the Purkinje cell expressed INPP5A and INPP4A. Multiple-tissue quantitative RT-PCR shows highest levels of Inpp5a in
the cerebellum and relatively low levels elsewhere (panel b). Expression was normalized to β-actin. Tissues were dissected from adult C57BL/6J mice (n=3). Immunofluorescence shows intense staining for INPP5A (green) in the adult C57BL/6J Purkinje cell layer (pcl) and their dendrites (ml) (panel c), but not in Inpp5a mutant animals. Sections were counterstained with DAPI (blue); scale bar indicates 50 μm. gcl granule cell layer
the first postnatal week, and pyramidal cells in the CA1 region of the hippocampus are lost in the third postnatal week. In comparison to other degenerative mutants, the Inpp4awbl mutant is unique in that Purkinje cells degenerate relatively early. The phenotype is due to a single base deletion in inositol polyphosphate 4-phosphatase type I [10]. The inositol 4-phosphatases, 4a and 4b, catalyze the removal of the 4-position phosphate from Ins(3,4)P2, Ins(1,3,4)P3, and PtdIns(3,4)P2 [11, 12]. Inpp4awbl mutants have abnormalities associated with endocytosis due to disruption of phosphatidylinositol (PtdIns) signaling [13, 14]. The mutation in Inpp4a also has a deleterious effect on enzyme activity toward the soluble inositol (Ins) substrates of this enzyme. In Purkinje cells, the enzymatic step immediately upstream of the type I 4-phosphatase is removal of the 5-position phosphate of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by INPP5A [15]. Interestingly, the only other 5-phosphatase (ptase) known to be expressed in the Purkinje cells is Inpp5e, which in vitro studies suggest that it only catalyzes reactions involving the PtdIns substrates [16]. Further, it has a subcellular localization that is restricted to the primary cilium [17]. This is not the case with the Inpp4a mutant, where no other 4-phosphatase is expressed in Purkinje cells. Thus, while we expect Inpp4a and Inpp5a mutant mice to have similar phenotypes, important differences may exist based on these small differences in their cellular functions.
In humans and mouse, mutations in type II 5-phosphatases have been associated with several disorders. Pertinent to the cerebellum, mutations in Inpp5e have been shown to cause vermis hypoplasia associated with Joubert syndrome [17, 18]. In contrast, the phenotypic consequences of a mutation in the type I 5-ptase, Inpp5a, were unknown. In this study, we show that deletion of Inpp5a causes a perinatal lethal phenotype in most mutant mice. A small percentage of mutants thrive and have a phenotype characterized by an ataxic gait and progressive Purkinje cell degeneration. The disease phenotype is consistent with the expression pattern of Inpp5a. Purkinje cell death is spatially patterned with surviving Purkinje cells appearing normal and maintaining molecular layer morphology. In addition, phosphatase activity in the mutant cerebellum is decreased toward soluble phosphoinositol substrates. A surprising difference between Inpp5a Gt(OST50073)Lex and Inpp4awbl phenotypes is that Purkinje cell degeneration has a later onset and is relatively slower in progression.
Materials and methods Animals All animals were bred and maintained under standard conditions at the University of Nebraska Medical Center research
Neurogenetics (2015) 16:277–285
vivarium in accordance with a protocol approved by the Animal Care and Use Committee at the University of Nebraska Medical Center (protocol numbers 04-085 and 10038). Mice were housed in microisolator cages and provided food and water ad libitum. The University of Nebraska Medical Center is in compliance with the NIH policy on the use of animals in research (Animal Welfare Act P.L. 89–544, as amended by P.L. 91–579 and P.L. 94–279) as well as the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23. The gene-trapped mice were obtained from the Texas Institute for Genomic Medicine as part of the NIH initiative (NOT-OD-06-012) purchasing 250 lines of gene-trapped mice from Lexicon Genetics. A mating cross of heterozygous B6.129S5-Inpp5a Gt(OST50073)Lex was established to generate mutant and control mice.
Genotyping DNA was isolated from tail tips excised from pups using a standard protocol [10]. All animal were genotyped blindly after visual phenotyping was performed. Genotyping for the Inpp5a mutation was performed using a multiplex polymerase chain reaction (PCR) reaction with allele-specific primers: 5′-TCCCTAGCATCAATGAGATACCA-3′, and 5′ACACCTACCTCACAAGGCTCAAGG-3′ resulting in a 101 bp wild-type product; and mutant, 5′-GATGCATCCA ACTTAGTGGC-3′ and 5′-ATAAACCCTCTTGCAGTTGC ATC-3′ resulting in a 182 bp mutant product. DNA was amplified by PCR using the following conditions: 40 ng of DNA in a 10 μl PCR reaction mixture containing 1.25 μl PCR buffer (100 mM Tris–HCl [pH 8.8], 500 mM KCl, 15 mM MgCl2, and 0.01 % w/v gelatin); 200 μM each dATP, dCTP, dGTP, and dTTP; 2.5 pmol of each forward and reverse primer; and 0.25 U Taq polymerase. Reaction mixtures were subjected to 40 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s.
Rotarod analysis To assess motor coordination, the age-matched heterozygous (n=7; 3 males, 4 females) and wild-type mice (n=10; 5 males, 5 females) were trained to walk on an accelerating rotarod at P37(Stoelting). During each of the three training days, the mice were placed on the rotarod at 6 rpm for 1 min, three sessions total with 5-min breaks between sessions. On the eighth day, P45, a test was performed that consisted of three trials of increasing speed, ranging from 4 to 40 rpm over a 5min period, was administered. The latency to fall off the rotarod was measured in seconds, and data were averaged per trial and statistically analyzed for significance using the Student’s t test.
279
Immunofluorescence for paraffin-embedded tissues Tissues were dissected from euthanized mutant and agematched littermates and fixed in either 4 % PFA in PBS or methanol:acetic acid (3:1). Tissues were paraffin-embedded in either sagittal or coronal planes and serially sectioned at a thickness of 10 μm. Sections were mounted on poly-L-lysine (Sigma)-coated slides. Immunofluorescence was performed using antibodies specific to CALB (Swant, 1:2,000), VGLUT2 (Chemicon, 1:200), INPP5A (Santa Cruz, 1:50), GFAP (Chemicon, 1:2,000), and INPP5E (Proteintech Group and Novus; similar results were obtained with both antibodies). Slide-mounted tissue sections were processed for immunofluorescence as before [19]. PFA-fixed section were boiled in 10 mM sodium citrate (pH 6.5) and allowed to cool to room temperature. Primary antibodies were incubated overnight 4 °C in 5 % horse serum blocking solution. Secondary antibodies (1:200 in blocking solution, AlexaFluor 488, 555, and 647, Invitrogen) were incubated on tissues for 2 h. Sections were counterstained with DAPI and visualized by fluorescent microscopy on a Ziess Axioplan 2 microscope. β-Galactosidase staining of tissue samples Fresh tissues were fixed at room temperature for 2 h with βgalactosidase fixative (0.2 % glutaraldehyde, 1.5 % formaldehyde, 5 mM EGTA, 2 mM MgCl2 in 100 mM sodium phosphate, pH 8.0), and washed three times for 30 min each in βgalactosidase wash buffer (2 mM MgCl2, 0.01 % sodium deoxycholate, 0.02 % Nonidet P-40 in 100 mM phosphate buffer, pH 8.0). Brain tissues were embedded in 4 % agarose and sectioned at 600 μm on Vibratome 1000 Sectioning System (The Vibratome Company). Adult brain sections, whole brains, and E13 whole embryos were incubated at 37 °C overnight in β-galactosidase staining solution (1 mg/ml Xgal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide in 100 mM sodium phosphate buffer, pH 8.0). The staining reaction was stopped by washing three times for 30 min each in PBS. Images were obtained with AF-S VR Micro-NIKKOR 105 mm f/2.8G IF-ED mounted on Nikon D90 DSLR camera (Nikon Inc.). Phosphatase activity assay Protein samples were extracted to measure phosphatase assay toward phosphoinositol and phosphatidylinositol substrates. Fresh cerebellum tissues from P0 mice were homogenized in 25 mM Tris, 250 mM sucrose, 500 mM KCl, 10 mM MgCl2, 2 mM EGTA, 1 mM DTT, pH 8.0 containing Complete Mini EDTA-Free Protease Inhibitor Cocktail (Roche Applied Sciences). Homogenates were centrifuged for 45 min at 13, 200 rpm at 4 °C, and supernatants were collected and
280
quantified using NanoDrop ND-1000 (NanoDrop Technologies). Protein (10 μg) were incubated with 1 μg of phosphoinositol or phosphatidylinositol substrate (Echelon Bioscience) and 100 μl of Malachite Green Solution in technical triplicate with and without the addition of phosphate substrates using Malachite Green K-1500 Phosphatase Assay (Echelon Biosciences). Production of free phosphate was measured using a Bio-Rad Model 680 Microplate Reader. Data were corrected for background activity using a no protein blank and statistically analyzed for significance using the Student’s t test. Western blot analysis was performed as before [19]. Multiple-tissue quantitative RT-PCR Quantitative real-time PCR (qRT-PCR) was used to determine the expression levels of Inpp5a relative to β-actin in multiple tissues from control animals. Total RNA was isolated from tissues of three euthanized wild-type mice using Trizol reagent (Invitrogen) per manufacturer’s instructions. The RNA was DNase treated with DNA-free (Ambion) to remove contaminating DNA, per manufacturer’s instructions. First-strand synthesis was performed using the RETROscript Kit (Ambion) on 2 μg of RNA template, per manufacturer’s instructions. Reaction products were diluted 1 to 100, and 1 μl was used as template for amplification using the following 10 μl reaction mixture: 100 pmol each forward and reverse primer and 6 μl Sybr green PCR master mix (ABI). Forward and reverse primers for Inpp5a and β-actin were chosen using Primer Express 3.0 (ABI). Quantitative RT-PCR was carried out on an ABI 7500 using the default cycling parameters. Reactions were performed in triplicate for the three separate RNA samples for wild type and mutant. As a measure of amplification, the number of cycles to a manually set ΔRn (dye fluorescence) threshold was determined as the ΔCt value (ABI 7500 System Software). ΔCt values were obtained for each sample, a total of nine for each gene (three triplicated replicates). Average ΔCt for each gene was calculated for each dataset as well as standard deviation and standard error. Significance was determined by the t test. The ΔΔCt was derived by comparison to a control gene, β-actin, for each sample. Comparisons between mutant and wild-type control samples were made for fold-change estimation using the comparative Ct method, 2−ΔΔCt, where ΔΔCt =ΔCttest gene-ΔCtβ-actin; the relative expression was calculated per 1,000 molecules of β-actin, 1000/2−ΔΔCt.
Results Inpp5a is expressed at high levels in Purkinje cells Cerebellar Purkinje cells express certain family members of inositol second messenger system genes at high levels, many
Neurogenetics (2015) 16:277–285
of which have been associated with ataxic movement disorders. Quantitative RT-PCR in a variety of adult tissues showed that of the 5-phosphatase family, Inpp5a was expressed at extremely high levels in the cerebellum (Fig. 1b), moderate levels in the cerebral cortex and muscle, and low levels elsewhere. Immunofluorescence using an INPP5A antibody showed expression restricted to Purkinje cells, with high staining in the dendritic arbors, and relatively less in the soma (Fig. 1c). Whole-mount X-gal staining of heterozygous embryos and adult brains from the Inpp5aGt(OST50073)Lex line (Fig. 2) confirmed that Inpp5a is highly expressed in the cerebellum in the adult (Fig. 2b). Low levels of staining were detected in the hippocampus and expression was not detected in any other neuroanatomical area (Fig. 2b). At embryonic day 13, E13, midline sagittal sections showed strong expression in the heart and tongue muscle in heterozygous animals (Fig. 2c,d). Deletion of Inpp5a causes ataxia and patterned Purkinje cell degeneration Heterozygous Inpp5aGt(OST50073)Lex mice were bred together to generate homozygous mutant animals. Significant in utero and perinatal loss was observed as less than 10 % of homozygous mutants survived past weaning. The gene trap inserted after exon 8, which truncates the protein after amino acid 215, effectively removes half of the protein including the predicted active site. RT-PCR using RNA isolated from a P0 mutant animal confirmed that the gene trap adversely affected the expression of wild-type Inpp5a (Fig. 2e). We were unable to detect a truncated protein by Western blot analysis as well; however, a small amount of normal protein was detected and probably the result of a small amount of splicing around the gene trap (Fig. 2f). Surviving homozygous mutant mice were smaller compared to littermate controls and had an ataxic gait detectable at P16 (Video S1). Although ataxia is not visually apparent in heterozygous animals, accelerating rotarod analysis revealed a deficit of motor ability. Heterozygous mice have an average of 55 % reduction in their ability to remain on a rotarod compared to wild-type mice at P45 (p < 0.001; Fig. 2g). Homozygous mice fell immediately off the rod and were therefore not included. Mutant cerebella were dissected and analyzed using immunofluorescence for the presence of neurodegeneration. At P20, the mutant cerebellum was generally smaller; however, only a few areas of Purkinje cell loss were prominent, most notable in lobes 8 and 11 (Fig. 3a,b). The mutant cerebellum had an average of 42 % reduction in cross-sectional area measured at the midline compared to heterozygous and wild-type littermates (n=3), this was approximately equal to the 30–40 % reduction in body weight due to runting that was observed in the mutant at this age. Defined regions of abnormally high levels of GFAP staining were detected in areas of Purkinje cell
Neurogenetics (2015) 16:277–285
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Fig. 2 Gene trap mutation disrupts the expression of Inpp5a. The gene trap insertion occurred between exons 8 and 9 of Inpp5a (panel a). β-gal-stained whole mount adult brains from heterozygous mice show high expression in the cerebellum, low expression in the hippocampus, and no expression elsewhere (panel b). Day 13.5 embryos were stained and sectioned in the sagittal plane at the midline, wildtype embryo showed no staining, while the heterozygote showed staining in the heart (arrow) and tongue (arrowhead) (panels c and d). The gene trap causes the elimination of the wild-type Inpp5a mRNA (panel e). A small amount of normal protein was detected in both the homozygote and heterozygote, probably due to the result of a small amount of splicing around the gene trap (panel f). Heterozygous mice have a significant reduction in their ability to remain on an accelerating rotarod compared to wild-type mice (p
ORIGINAL ARTICLE
Deletion of Inpp5a causes ataxia and cerebellar degeneration in mice Andy W. Yang 1 & Andrew J. Sachs 1 & Arne M. Nystuen 1
Received: 30 March 2015 / Accepted: 5 May 2015 / Published online: 9 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The progressive and permanent loss of cerebellar Purkinje cells (PC) is a hallmark of many inherited ataxias. Mutations in several genes involved in the regulation of Ca2+ release from intracellular stores by the second messenger IP3 have been associated with PC dysfunction or death. While much is known about the defects in production and response to IP3, less is known about the defects in breakdown of the IP3 second messenger. A mutation in Inpp4a of the pathway is associated with a severe, early-onset PC degeneration in the mouse model weeble. The step preceding the removal of the 4-phosphate is the removal of the 5-phosphate by Inpp5a. Gene expression analysis was performed on an Inpp5aGt(OST50073)Lex mouse generated by gene trap insertion using quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blot. Phenotypic analyses were performed using rotarod, β-galactosidase staining, and phosphatase activity assay. Statistical significance was calculated. The deletion of Inpp5a causes an early-onset yet slowly progressive PC degeneration and ataxia. Homozygous mutants (90 %) exhibit perinatal lethality; surviving homozygotes show locomotor instability at P16. A consistent pattern of PC loss in the cerebellum is initially detectable by weaning and widespread by P60. Phosphatase activity toward
Electronic supplementary material The online version of this article (doi:10.1007/s10048-015-0450-4) contains supplementary material, which is available to authorized users. * Andy W. Yang [email protected] Andrew J. Sachs [email protected] Arne M. Nystuen [email protected] 1
The Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA
phosphoinositol substrates is reduced in the mutant relative to littermates. The ataxic phenotype and characteristics neurodegeneration of the Inpp5aGt(OST50073)Lex mouse indicate a crucial role for Inpp5a in PC survival. The identification of the molecular basis of the selective PC survival will be important in defining a neuroprotective gene applicable to establishing a disease mechanism. Keywords Inpp5a . Knockout mouse . Cerebellar degeneration . Ataxia
Introduction In neurons, the central mechanism controlling calcium release from intracellular stores involves the generation of a second messenger, inositol 1,4,5-trisphosphate (IP3), in response to an extracellular signal [1]. There are multiple steps in this pathway, which are performed by proteins that are encoded by large gene families [2]. In cerebellar Purkinje cells, an excitatory glutamate signal from climbing fibers is transduced through metabotropic glutamate receptor 1 (mGluR1) [3, 4]. This causes the generation of IP3 via guanine nucleotidebinding protein, alpha q polypeptide (GαQ) [5], and phospholipase C beta 4 (PLCβ4) [6]. IP3 binds inositol 1,4,5-trisphosphate receptor 1 (ITPR1) and causes the release of Ca2+ (Fig. 1a) [7]. The above genes, involved in the generation and response to IP3, have been associated with neurological disorders in human and mouse [8, 9]; however, relatively less is known about perturbations in the breakdown of the IP3 signal. One such model, Inpp4awbl, is a recessive mouse mutant characterized by severe ataxia and pre-wean lethality. The Inpp4awbl mutant has a unique combination of neuroanatomical abnormalities including neurodegeneration in the cerebellum and hippocampus. Specifically, Purkinje cells are lost in
278
Neurogenetics (2015) 16:277–285
Fig. 1 Inpp5a is expressed in the cerebellar Purkinje cells and muscle. The pathway resulting in the release of Ca2+ from intracellular stores is shown in panel a, with the members of the pathway expressed in Purkinje cells. Phosphate groups (red dots) are removed from the inositol backbone by enzymes including the Purkinje cell expressed INPP5A and INPP4A. Multiple-tissue quantitative RT-PCR shows highest levels of Inpp5a in
the cerebellum and relatively low levels elsewhere (panel b). Expression was normalized to β-actin. Tissues were dissected from adult C57BL/6J mice (n=3). Immunofluorescence shows intense staining for INPP5A (green) in the adult C57BL/6J Purkinje cell layer (pcl) and their dendrites (ml) (panel c), but not in Inpp5a mutant animals. Sections were counterstained with DAPI (blue); scale bar indicates 50 μm. gcl granule cell layer
the first postnatal week, and pyramidal cells in the CA1 region of the hippocampus are lost in the third postnatal week. In comparison to other degenerative mutants, the Inpp4awbl mutant is unique in that Purkinje cells degenerate relatively early. The phenotype is due to a single base deletion in inositol polyphosphate 4-phosphatase type I [10]. The inositol 4-phosphatases, 4a and 4b, catalyze the removal of the 4-position phosphate from Ins(3,4)P2, Ins(1,3,4)P3, and PtdIns(3,4)P2 [11, 12]. Inpp4awbl mutants have abnormalities associated with endocytosis due to disruption of phosphatidylinositol (PtdIns) signaling [13, 14]. The mutation in Inpp4a also has a deleterious effect on enzyme activity toward the soluble inositol (Ins) substrates of this enzyme. In Purkinje cells, the enzymatic step immediately upstream of the type I 4-phosphatase is removal of the 5-position phosphate of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by INPP5A [15]. Interestingly, the only other 5-phosphatase (ptase) known to be expressed in the Purkinje cells is Inpp5e, which in vitro studies suggest that it only catalyzes reactions involving the PtdIns substrates [16]. Further, it has a subcellular localization that is restricted to the primary cilium [17]. This is not the case with the Inpp4a mutant, where no other 4-phosphatase is expressed in Purkinje cells. Thus, while we expect Inpp4a and Inpp5a mutant mice to have similar phenotypes, important differences may exist based on these small differences in their cellular functions.
In humans and mouse, mutations in type II 5-phosphatases have been associated with several disorders. Pertinent to the cerebellum, mutations in Inpp5e have been shown to cause vermis hypoplasia associated with Joubert syndrome [17, 18]. In contrast, the phenotypic consequences of a mutation in the type I 5-ptase, Inpp5a, were unknown. In this study, we show that deletion of Inpp5a causes a perinatal lethal phenotype in most mutant mice. A small percentage of mutants thrive and have a phenotype characterized by an ataxic gait and progressive Purkinje cell degeneration. The disease phenotype is consistent with the expression pattern of Inpp5a. Purkinje cell death is spatially patterned with surviving Purkinje cells appearing normal and maintaining molecular layer morphology. In addition, phosphatase activity in the mutant cerebellum is decreased toward soluble phosphoinositol substrates. A surprising difference between Inpp5a Gt(OST50073)Lex and Inpp4awbl phenotypes is that Purkinje cell degeneration has a later onset and is relatively slower in progression.
Materials and methods Animals All animals were bred and maintained under standard conditions at the University of Nebraska Medical Center research
Neurogenetics (2015) 16:277–285
vivarium in accordance with a protocol approved by the Animal Care and Use Committee at the University of Nebraska Medical Center (protocol numbers 04-085 and 10038). Mice were housed in microisolator cages and provided food and water ad libitum. The University of Nebraska Medical Center is in compliance with the NIH policy on the use of animals in research (Animal Welfare Act P.L. 89–544, as amended by P.L. 91–579 and P.L. 94–279) as well as the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23. The gene-trapped mice were obtained from the Texas Institute for Genomic Medicine as part of the NIH initiative (NOT-OD-06-012) purchasing 250 lines of gene-trapped mice from Lexicon Genetics. A mating cross of heterozygous B6.129S5-Inpp5a Gt(OST50073)Lex was established to generate mutant and control mice.
Genotyping DNA was isolated from tail tips excised from pups using a standard protocol [10]. All animal were genotyped blindly after visual phenotyping was performed. Genotyping for the Inpp5a mutation was performed using a multiplex polymerase chain reaction (PCR) reaction with allele-specific primers: 5′-TCCCTAGCATCAATGAGATACCA-3′, and 5′ACACCTACCTCACAAGGCTCAAGG-3′ resulting in a 101 bp wild-type product; and mutant, 5′-GATGCATCCA ACTTAGTGGC-3′ and 5′-ATAAACCCTCTTGCAGTTGC ATC-3′ resulting in a 182 bp mutant product. DNA was amplified by PCR using the following conditions: 40 ng of DNA in a 10 μl PCR reaction mixture containing 1.25 μl PCR buffer (100 mM Tris–HCl [pH 8.8], 500 mM KCl, 15 mM MgCl2, and 0.01 % w/v gelatin); 200 μM each dATP, dCTP, dGTP, and dTTP; 2.5 pmol of each forward and reverse primer; and 0.25 U Taq polymerase. Reaction mixtures were subjected to 40 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s.
Rotarod analysis To assess motor coordination, the age-matched heterozygous (n=7; 3 males, 4 females) and wild-type mice (n=10; 5 males, 5 females) were trained to walk on an accelerating rotarod at P37(Stoelting). During each of the three training days, the mice were placed on the rotarod at 6 rpm for 1 min, three sessions total with 5-min breaks between sessions. On the eighth day, P45, a test was performed that consisted of three trials of increasing speed, ranging from 4 to 40 rpm over a 5min period, was administered. The latency to fall off the rotarod was measured in seconds, and data were averaged per trial and statistically analyzed for significance using the Student’s t test.
279
Immunofluorescence for paraffin-embedded tissues Tissues were dissected from euthanized mutant and agematched littermates and fixed in either 4 % PFA in PBS or methanol:acetic acid (3:1). Tissues were paraffin-embedded in either sagittal or coronal planes and serially sectioned at a thickness of 10 μm. Sections were mounted on poly-L-lysine (Sigma)-coated slides. Immunofluorescence was performed using antibodies specific to CALB (Swant, 1:2,000), VGLUT2 (Chemicon, 1:200), INPP5A (Santa Cruz, 1:50), GFAP (Chemicon, 1:2,000), and INPP5E (Proteintech Group and Novus; similar results were obtained with both antibodies). Slide-mounted tissue sections were processed for immunofluorescence as before [19]. PFA-fixed section were boiled in 10 mM sodium citrate (pH 6.5) and allowed to cool to room temperature. Primary antibodies were incubated overnight 4 °C in 5 % horse serum blocking solution. Secondary antibodies (1:200 in blocking solution, AlexaFluor 488, 555, and 647, Invitrogen) were incubated on tissues for 2 h. Sections were counterstained with DAPI and visualized by fluorescent microscopy on a Ziess Axioplan 2 microscope. β-Galactosidase staining of tissue samples Fresh tissues were fixed at room temperature for 2 h with βgalactosidase fixative (0.2 % glutaraldehyde, 1.5 % formaldehyde, 5 mM EGTA, 2 mM MgCl2 in 100 mM sodium phosphate, pH 8.0), and washed three times for 30 min each in βgalactosidase wash buffer (2 mM MgCl2, 0.01 % sodium deoxycholate, 0.02 % Nonidet P-40 in 100 mM phosphate buffer, pH 8.0). Brain tissues were embedded in 4 % agarose and sectioned at 600 μm on Vibratome 1000 Sectioning System (The Vibratome Company). Adult brain sections, whole brains, and E13 whole embryos were incubated at 37 °C overnight in β-galactosidase staining solution (1 mg/ml Xgal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide in 100 mM sodium phosphate buffer, pH 8.0). The staining reaction was stopped by washing three times for 30 min each in PBS. Images were obtained with AF-S VR Micro-NIKKOR 105 mm f/2.8G IF-ED mounted on Nikon D90 DSLR camera (Nikon Inc.). Phosphatase activity assay Protein samples were extracted to measure phosphatase assay toward phosphoinositol and phosphatidylinositol substrates. Fresh cerebellum tissues from P0 mice were homogenized in 25 mM Tris, 250 mM sucrose, 500 mM KCl, 10 mM MgCl2, 2 mM EGTA, 1 mM DTT, pH 8.0 containing Complete Mini EDTA-Free Protease Inhibitor Cocktail (Roche Applied Sciences). Homogenates were centrifuged for 45 min at 13, 200 rpm at 4 °C, and supernatants were collected and
280
quantified using NanoDrop ND-1000 (NanoDrop Technologies). Protein (10 μg) were incubated with 1 μg of phosphoinositol or phosphatidylinositol substrate (Echelon Bioscience) and 100 μl of Malachite Green Solution in technical triplicate with and without the addition of phosphate substrates using Malachite Green K-1500 Phosphatase Assay (Echelon Biosciences). Production of free phosphate was measured using a Bio-Rad Model 680 Microplate Reader. Data were corrected for background activity using a no protein blank and statistically analyzed for significance using the Student’s t test. Western blot analysis was performed as before [19]. Multiple-tissue quantitative RT-PCR Quantitative real-time PCR (qRT-PCR) was used to determine the expression levels of Inpp5a relative to β-actin in multiple tissues from control animals. Total RNA was isolated from tissues of three euthanized wild-type mice using Trizol reagent (Invitrogen) per manufacturer’s instructions. The RNA was DNase treated with DNA-free (Ambion) to remove contaminating DNA, per manufacturer’s instructions. First-strand synthesis was performed using the RETROscript Kit (Ambion) on 2 μg of RNA template, per manufacturer’s instructions. Reaction products were diluted 1 to 100, and 1 μl was used as template for amplification using the following 10 μl reaction mixture: 100 pmol each forward and reverse primer and 6 μl Sybr green PCR master mix (ABI). Forward and reverse primers for Inpp5a and β-actin were chosen using Primer Express 3.0 (ABI). Quantitative RT-PCR was carried out on an ABI 7500 using the default cycling parameters. Reactions were performed in triplicate for the three separate RNA samples for wild type and mutant. As a measure of amplification, the number of cycles to a manually set ΔRn (dye fluorescence) threshold was determined as the ΔCt value (ABI 7500 System Software). ΔCt values were obtained for each sample, a total of nine for each gene (three triplicated replicates). Average ΔCt for each gene was calculated for each dataset as well as standard deviation and standard error. Significance was determined by the t test. The ΔΔCt was derived by comparison to a control gene, β-actin, for each sample. Comparisons between mutant and wild-type control samples were made for fold-change estimation using the comparative Ct method, 2−ΔΔCt, where ΔΔCt =ΔCttest gene-ΔCtβ-actin; the relative expression was calculated per 1,000 molecules of β-actin, 1000/2−ΔΔCt.
Results Inpp5a is expressed at high levels in Purkinje cells Cerebellar Purkinje cells express certain family members of inositol second messenger system genes at high levels, many
Neurogenetics (2015) 16:277–285
of which have been associated with ataxic movement disorders. Quantitative RT-PCR in a variety of adult tissues showed that of the 5-phosphatase family, Inpp5a was expressed at extremely high levels in the cerebellum (Fig. 1b), moderate levels in the cerebral cortex and muscle, and low levels elsewhere. Immunofluorescence using an INPP5A antibody showed expression restricted to Purkinje cells, with high staining in the dendritic arbors, and relatively less in the soma (Fig. 1c). Whole-mount X-gal staining of heterozygous embryos and adult brains from the Inpp5aGt(OST50073)Lex line (Fig. 2) confirmed that Inpp5a is highly expressed in the cerebellum in the adult (Fig. 2b). Low levels of staining were detected in the hippocampus and expression was not detected in any other neuroanatomical area (Fig. 2b). At embryonic day 13, E13, midline sagittal sections showed strong expression in the heart and tongue muscle in heterozygous animals (Fig. 2c,d). Deletion of Inpp5a causes ataxia and patterned Purkinje cell degeneration Heterozygous Inpp5aGt(OST50073)Lex mice were bred together to generate homozygous mutant animals. Significant in utero and perinatal loss was observed as less than 10 % of homozygous mutants survived past weaning. The gene trap inserted after exon 8, which truncates the protein after amino acid 215, effectively removes half of the protein including the predicted active site. RT-PCR using RNA isolated from a P0 mutant animal confirmed that the gene trap adversely affected the expression of wild-type Inpp5a (Fig. 2e). We were unable to detect a truncated protein by Western blot analysis as well; however, a small amount of normal protein was detected and probably the result of a small amount of splicing around the gene trap (Fig. 2f). Surviving homozygous mutant mice were smaller compared to littermate controls and had an ataxic gait detectable at P16 (Video S1). Although ataxia is not visually apparent in heterozygous animals, accelerating rotarod analysis revealed a deficit of motor ability. Heterozygous mice have an average of 55 % reduction in their ability to remain on a rotarod compared to wild-type mice at P45 (p < 0.001; Fig. 2g). Homozygous mice fell immediately off the rod and were therefore not included. Mutant cerebella were dissected and analyzed using immunofluorescence for the presence of neurodegeneration. At P20, the mutant cerebellum was generally smaller; however, only a few areas of Purkinje cell loss were prominent, most notable in lobes 8 and 11 (Fig. 3a,b). The mutant cerebellum had an average of 42 % reduction in cross-sectional area measured at the midline compared to heterozygous and wild-type littermates (n=3), this was approximately equal to the 30–40 % reduction in body weight due to runting that was observed in the mutant at this age. Defined regions of abnormally high levels of GFAP staining were detected in areas of Purkinje cell
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Fig. 2 Gene trap mutation disrupts the expression of Inpp5a. The gene trap insertion occurred between exons 8 and 9 of Inpp5a (panel a). β-gal-stained whole mount adult brains from heterozygous mice show high expression in the cerebellum, low expression in the hippocampus, and no expression elsewhere (panel b). Day 13.5 embryos were stained and sectioned in the sagittal plane at the midline, wildtype embryo showed no staining, while the heterozygote showed staining in the heart (arrow) and tongue (arrowhead) (panels c and d). The gene trap causes the elimination of the wild-type Inpp5a mRNA (panel e). A small amount of normal protein was detected in both the homozygote and heterozygote, probably due to the result of a small amount of splicing around the gene trap (panel f). Heterozygous mice have a significant reduction in their ability to remain on an accelerating rotarod compared to wild-type mice (p
