brain research 1632 (2016) 141–155
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Research Report Mice null for NEDD9 (HEF1) display extensive hippocampal dendritic spine loss and cognitive impairment
n D.C. Knutsona, A.M. Mitzeya, L.E. Taltonb, M. Clagett-Damea,c, aDepartment of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706, USA bBehavioral Testing Core Facility, University of California, Los Angeles, CA 90095, USA cPharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, WI 53705, USA article info abstract
Article history: NEDD9 (neural precursor cell expressed, developmentally down-regulated 9) is a member Accepted 1 December 2015 of the CAS (Crk-associated substrate) family of scaffolding proteins that regulate cell Available online 10 December 2015 adhesion and migration. A Nedd9 knock-out/lacZ knock-in mouse (Nedd9 / ) was devel- oped in order to study Nedd9 expression and function in the nervous system. Herein we Keywords: show that NEDD9 is expressed in the adult brain and is prominently expressed in the NEDD9 hippocampus. Behavioral testing uncovered functional deficits in Nedd9 / mice. In the Retinoic acid-induced Morris water maze test, Nedd9 / mice showed deficits in both the ability to learn the task differentiation as well as in their ability to recall the platform location. There was no change in the gross Hippocampus morphology of the hippocampus, and stereological analysis of BrdU-labeled newly formed Dendritic spine density hippocampal cells suggested that this defect is not secondary to altered neurogenesis. CAS However, analysis of the hippocampus revealed extensive loss of dendritic spine density in Null mutant both the dentate gyrus (DG) and CA1 regions. Spine loss occurred equally across all branch orders and regions of the dendrite. Analysis of spine density in Nedd9 / mice at 1.5, 6 and 10 months revealed an age-dependent spine loss. This work shows that NEDD9 is required for the maintenance of dendritic spines in the hippocampus, and suggests it could play a role in learning and memory. & 2015 Elsevier B.V. All rights reserved.
Abbreviations: ABL, Abelson murine leukemia; ANOVA, analysis of variance; ATN, anterior group of the dorsal thalamus; AAALAC, Association for the Assessment and Accreditation of Laboratory Animal Care International; BLA, basolateral amygdalar nucleus; BST, bed nuclei of the stria terminalis; BCAR1, breast cancer anti-estrogen resistance 1; BrdU, bromodeoxyuridine; CASS4, CAS scaffolding protein family member 4; CP, caudoputamen; CBN, cerebellar nuclei; CN, cochlear nuclei; C.E., coefficient of error; CA1, cornu ammonis 1; CA1sp, cornu ammonis 1 pyramidal area; CA3, cornu ammonis 3; CA3sp, cornu ammonis 3 pyramidal area; CA1sr, cornu ammonis 1 stratum radiatum; COA, cortical amygdalar area; Cre, Cre recombinase; CAS, Crk- associated substrate; DG, dentate gyrus; DGsg, dentate gyrus granule cell layer; DR, dorsal nucleus raphe; EFS/Sin, embryonal Fyn substrate/Src-interacting; ES, embryonic stem; EP, endopiriform nucleus; VII, facial motor nucleus; FAK, focal adhesion kinase; þ GCL, granule cell layer; HET, Nedd9 / , heterozygous; H, hilus; HEF1, human enhancer of filamentation 1; HY, hypothalamus; isl, islands of Calleja; CTX, isocortex; KO, knock-out, Nedd9 / ; LSN, lateral septal nucleus; LOAD, late-onset Alzheimer’s disease; http://dx.doi.org/10.1016/j.brainres.2015.12.005 0006-8993/& 2015 Elsevier B.V. All rights reserved. 142 brain research 1632 (2016) 141–155
treatment with a Rho kinase inhibitor (Bargon et al., 2005). 1. Introduction Knockdown of Nedd9 reduces neural crest cell migration in the chick embryo (Aquino et al., 2009). More recently, single NEDD9 (HEF1/CAS-L/CASS2) is a member of the CAS family of nucleotide polymorphisms (SNPs) in Nedd9 have been linked multidomain docking or scaffolding proteins that includes to pathological conditions including late-onset Alzheimer’s CAS BCAR1/p130 , EFS/SIN and CASS4/HEPL (Guerrero et al., (LOAD) and Parkinson’s disease (Beck et al., 2014; Fu et al., 2012; Nikonova et al., 2014; Tikhmyanova et al., 2010). Nedd9 2012; Li et al., 2008; Tedde et al., 2010; Xing et al., 2011). fi fi was rst identi ed as a developmentally down-regulated However, where Nedd9 is found in the adult brain and gene in mouse brain (Kumar et al., 1992), and was later whether it plays a role in normal nervous system function fi independently identi ed as a human protein that enhanced is not well understood. pseudohyphal budding in yeast (HEF1; Law et al., 1996) and as In this report, we generate a mouse null for Nedd9 that also a protein expressed in lymphocytes (CAS-L; Minegishi et al., contains a knock-in of lacZ in order to examine the expres- fi fi 1996) with similarity to the rst identi ed CAS family mem- sion and function of NEDD9 in the adult nervous system. CAS ber, p130 (Reynolds et al., 1989; Sakai et al., 1994). EFS/Sin NEDD9/LacZ is highly expressed in the adult hippocampus, was initially described as a Fyn SH3-domain-binding protein and Nedd9 / null mutant mice show a deficit in learning and (Alexandropoulos and Baltimore, 1996; Ishino et al., 1995), memory along with a dramatic loss of dendritic spines, and the most recently described CAS family member, CASS4, providing evidence of a new function for NEDD9 in nervous has many similar activities to NEDD9 in vitro (Singh et al., system maintenance. 2008). CAS family members act downstream of cell surface receptors and cytoplasmic protein tyrosine kinases (FAK, SFK, and ABL) and function as platforms for the assembly of 2. Results multiprotein complexes to regulate cytoskeletal dynamics. Early studies described NEDD9 as a signaling intermediate in 2.1. Generation of the Nedd9 knock-out (KO)/lacZ knock-in pathways affecting cellular attachment to the extracellular (Nedd9 / ) mouse matrix and cellular motility in lymphoid and epithelial cells (Fashena et al., 2002; Law et al., 1998; Ohashi et al., 1999; van To generate Nedd9 null mutant mice (Nedd9 / ), the Nedd9 Seventer et al., 2001). Our lab identified Nedd9 in a screen for genomic region containing exon 3 of the NEDD9 protein was all-trans retinoic acid responsive genes in a neuroblastoma replaced with lacZ and a Neo resistance cassette by homo- cell line that extends neurites and shows changes in adhesive logous recombination in embryonic stem (ES) cells. The Nedd9 properties in response to retinoic acid (Merrill et al., 2004a, gene has two promoters and two translational start sites 2004b). (found in exons 2A and 2B) that produce two nearly identical Although much of what is currently known about Nedd9 isoforms of the protein differing only in the first three amino comes from studies of its role in cancer cell metastasis and acids (Sasaki et al., 2005). LacZ was introduced into exon 3, the lymphocyte migration, evidence is emerging that it also first exon common to both transcripts, putting lacZ in frame functions in the nervous system. Nedd9 mRNA is expressed with either of the two Nedd9 translational start sites to in mouse brain, and is also found in the developing nervous generate a fusion protein composed of the first 5 amino acids system of the rat embryo (Kumar et al., 1992; Merrill et al., of the NEDD9 protein and 1 foreign amino acid followed by 2004b). Mouse embryos transgenic for a 5.2 kb region of the lacZ sequence (Fig. 1A). This disrupted the protein coding Nedd9 promoter express high levels of Nedd9 in the caudal sequence of Nedd9 and inserted a stop codon and a poly- hindbrain neuroepithelium, spinal cord, dorsal root ganglia adenylation signal into exon 3 of the gene in order to and migrating neural crest, and expression is dependent terminate transcription prior to reaching the remaining Nedd9 upon an intact retinoic acid response element (Knutson and coding exons (exons 4–8) and allows for lacZ expression Clagett-Dame, 2008, 2015). Nedd9 is expressed in neural driven by the Nedd9 promotor. Correct integration of the progenitor and progenitor-like cells. It is induced in neuro- targeting vector was identified by PCR and Southern blot blastoma cells undergoing differentiation by retinoic acid analysis (Fig. 1B). The mutant loci were confirmed by PCR (Merrill et al., 2004a, 2004b), and neurite length and number screening of tail-derived genomic DNA (Fig. 1C), and two is enhanced in PC12 cells overexpressing Nedd9 (Sasaki et al., females and one male heterozygous (HET) mouse from the 2005). The formation of neurites in a non-neuronal cell type same ES line were generated. Western blot analysis showed a has also been reported with Nedd9 overexpression and complete loss of NEDD9 protein in the null mutant (Fig. 1D).
(footnote continued) MB, midbrain; ML, molecular layer; V, motor nucleus of the trigeminal; NEDD9, neural precursor cell expressed, developmentally down-regulated 9; Neo, neomycin; ACB, nucleus accumbens; PB, parabrachial nucleus; PFA, paraformaldehyde; PGK, phosphoglycerine kinase; PG, pontine gray; PCL, Purkinje cell layer region; SGZ, subgranular zone; PSD, postsynaptic density; RHP, retrohippocampal region; RSP, retrosplenial area; SNPs, single nucleotide polymorphisms; SPV, spinal nucleus of the trigeminal; SFK, SRC family kinase; SOC, superior olivary complex; TRN, tegmental reticular nucleus; TH, thalamus; þ þ VNC, vestibular nuclei; WT, wild-type, Nedd9 / n Corresponding author at: Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA. Fax: þ1 608 262 7122. E-mail address: [email protected] (M. Clagett-Dame). brain research 1632 (2016) 141–155 143
2.2. Deletion of Nedd9 does not affect growth and 2.3. Nedd9 is highly expressed in the adult hippocampus reproduction β-galactosidase (LacZ) staining was examined in thick coronal Nedd9 KO/lacZ knock-in animals grew normally, were repro- (Fig. 2A–F, right half) and sagittal (Supplementary Fig. 1A–E) ductively active, survived up to 2.5 years of age, and were sections of Nedd9 KO/lacZ knock-ins to determine the location of indistinguishable from wild-type littermates in agreement NEDD9 expression in the brain. β-galactosidase staining was / with the results of Seo et al. (2005) in CAS-L-deficient mice. At observed in several regions of the adult brain in Nedd9 necropsy, there was no evidence of any abnormality in the mutants with particularly pronounced expression in multiple orientation/placement of major organs in Nedd9 / mice layers of the cerebral cortex and hippocampal formation. NEDD9/LacZ staining was observed in the hippocampus proper, (data not shown). with most prominent expression in the pyramidal area of the 144 brain research 1632 (2016) 141–155
CA1 and CA3 fields (CA1sp and CA3sp, respectively), and in the were examined as a control, and no LacZ staining was observed granule cell layer of the dentate gyrus (DGsg) (Fig. 2CandD; in these sections (Fig. 2A–F, left half). Supplementary Fig. 1A, B and D). The retrohippocampus (RHP), and the retrosplenial cortex (RSP) were also stained (Fig. 2Cand 2.4. Nedd9 / mice show deficits in a spatial learning D; Supplementary Fig. 1 A and D), and expression was observed task throughout multiple layers of the isocortex (CTX or neocortex) (Fig. 2A–E; Supplementary Fig. 1A and D). Two additional sites of The expression of NEDD9 in the brain prompted us to test expression included the anterior group of the dorsal thalamus whether deletion of Nedd9 led to any behavioral alterations. fi (ATN; Supplementary Fig. 1A)andthelateralseptalnucleus Mice rst underwent a primary SHIRPA screen designed to (LSN; Fig. 2B). Other regions of lesser staining included the test muscle, cerebellar, sensory and neuropsychiatric func- / endopiriform nucleus (EP; Fig. 2A–D), bed nuclei of the stria tion (Irwin, 1968; Rogers et al., 1997). The Nedd9 mutants fi þ/þ terminalis (BST, Supplementary Fig. 1A), cortical amygdalar area did not differ signi cantly from the Nedd9 animals in any (COA; Fig. 2B–D), nucleus accumbens (ACB; Fig. 2A; Supplemen- of the 42 SHIRPA tasks and the performance of both groups was within the range considered normal for the C57BL/6 tary Fig. 1A), islands of Calleja of the olfactory tubercle (isl; strain (data not shown). Thus, KO of the Nedd9 gene had no Fig. 2A) the basolateral amygdalar nucleus (BLA; Fig. 2D) and the gross effect on sensory modalities of vision, audition, whisker caudoputamen (CP; Fig. 2B; Supplementary Fig. 1A and D). There or vestibular function or other attributes such as aggression, was also light or diffuse staining in the hypothalamus (HY; gait, heart rate, body or limb tone, muscle strength, respira- Fig. 2D; Supplementary Fig. 1A) and midbrain (MB; Supplemen- tion, or pain sensitivity. tary Fig. 1A), including the dorsal nucleus raphe (DR, Fig. 2E). In Because the NEDD9–LacZ fusion protein was highly the hindbrain, staining in the pons occurred in the parabrachial expressed in the hippocampus, mice were trained and tested nucleus (PB; Supplementary Fig. 1C), tegmental reticular nucleus in the hidden platform version of the Morris water maze, a (TRN; Fig. 2E), pontine gray (PG; Supplementary Fig. 1AandC), test in which the hippocampus is known to support spatial superior olivary complex (SOC; Supplementary Fig. 1AandC), processing demands. In this test, the animal learns the and the motor nucleus of the trigeminal (V; Supplementary location of the escape platform in relation to the cues in Fig. 1C). Medullary expression included the vestibular nuclei the periphery of the room. The training (T) and probe (VNC; Supplementary Fig. 1A), facial motor nucleus (VII; Supple- (P) testing schedule is depicted in Fig. 3A. In the training mentary Fig. 1A and C), lateral reticular nucleus (LRN; Supple- portion, wild-type (WT) mice showed more rapid learning of mentary Fig. 1A and C), cochlear nuclei (CN; Supplementary the task compared to mutants, with a significantly shorter Fig. 1D), and the spinal nucleus of the trigeminal (SPV; Fig. 2F; latency to find the platform in bin 2. The latency to find the Supplementary Fig. 1D). In the cerebellum, expression was seen platform improved with training in WT, and this group in the cerebellar nuclei (CBN; Fig. 2F; Supplementary Fig. 1Aand showed significantly shorter latencies in bins 6 and 7 of the D), as well as between the granule and molecular cell layers of test compared to Nedd9 / mice (Fig. 3B). the folia, a region in which the Purkinje cells are located (PCL; After training sessions 10, 16, 22, and 28, spatial learning was þ þ Fig. 2F; Supplementary Fig. 1AandE).BrainsfromNedd9 / mice assessed using probe trials (P1, P2, P3, and P4) in which the
Fig. 1 – Generation of Nedd9 KO/lacZ knock-in mice. Schematic of the Nedd9 genomic locus (A) and the location of the 50 (pink) and 30 (purple) homology arms in the targeting vector. The targeted KO/knock-in (lacZ, light blue) allele before and after Cre recombination is also depicted. Dotted lines indicate the intronic regions that flank exon 3, these comprise the homology arms used in the targeting vector. The approximate size and locations of exons 3–6 are depicted by black rectangles labeled beneath with the exon number. Restriction sites are listed. The 50 (50, red box), 30 (30, green box) and Neo (Neo, dark blue) probes are indicated, as well as location of genotyping PCR primers (P1–P3, gray arrows). Expected sizes of restriction fragments detected by probes used are shown below the appropriate sequence (red, green and dark blue arrow-headed lines). LoxP sites flank the Neo selection gene (PGK Neo) allowing for later deletion by mating with Cre expressing mice. Analysis of target gene integration by Southern blotting, PCR and Western blotting to verify loss of both Nedd9 mRNA and protein in the null mutant mice (B–D). Southern blots (B) show the expected integration of the targeting construct. DNA was isolated from expanded targeted ES cells and from WT cells (positive probe control was included but is not shown). Top panel: DNA digested with ScaI and hybridized with the 50-probe shows expected WT band (27.7kb) and targeted KO/knock-in allele (KO band, 15.3 kb) bands in HET ES cells. Bottom panel: DNA digested with XbaI and hybridized with the 30-probe shows expected WT band (10.0 kb) and targeted KO/knock-in allele (KO band, 8.2 kb) bands in HET ES cells. PCR analysis of tail snip DNA þ þ þ (C) from Nedd9 / (WT), Nedd9 / (HET), and Nedd9 / (KO) adult male mice. Primers P1, P2, and P3 (shown in A) were combined. WT band: 753 bp and KO/knock-in band (KO band): 334 bp. Western blot analysis (D) indicates the complete loss of þ þ endogenous NEDD9 protein expression in Nedd9 / animals. Protein-containing lysates prepared from Nedd9 / (WT), þ Nedd9 / (HET), and Nedd9 / (KO) adult lungs were resolved on a SDS polyacrylamide gel. Lung was chosen for analysis as it was enriched in the NEDD9 protein enabling definitive detection on the Western blot. The blot was cut in half and the upper half was probed with a rabbit anti-NEDD9 antibody that detected the expected 105 kDa (phosphorylated) and 115 kDa þ þ þ (hyperphosphorylated) bands (Law et al., 1998) in the Nedd9 / and Nedd9 / but not the Nedd9 / sample. As a loading control, the bottom half was probed with mouse anti-β-actin clone AC-15 antibody that detected the expected 42 kDa band in all samples. brain research 1632 (2016) 141–155 145
Fig. 2 – NEDD9 is highly expressed in the hippocampus of the adult mouse brain. NEDD9–LacZ fusion protein expression was mapped by β-galactosidase staining of coronal brain slices. Images are arranged from rostral (anterior) to caudal (posterior), with the cartoon at top (modified from Sunkin et al., (2013)) showing the relative position of each section. For each section (A– þ þ F), the left hemisphere from a Nedd9 / mouse is shown as a control and the right hemisphere is a representative Nedd9 / section from a mouse with lacZ knocked into both alleles. Sections were counterstained with nuclear fast red. Labeled areas indicate regions positive for β-galactosidase stain (blue). Abbreviations: BLA, basolateral amygdalar nucleus; CA1, cornu ammonis 1; CA3, cornu ammonis 3; CP, caudoputamen; CBN, cerebellar nuclei; CN, cochlear nucleus; COA, cortical amygdalar area; DG, dentate gyrus; DR, dorsal nucleus raphe; EP, endopiriform nucleus; VII, facial motor nucleus; HPF, hippocampal formation; HY, hypothalamus; isl, islands of Calleja; CTX, isocortex; LSN, lateral septal nucleus; ACB, nucleus accumbens; PCL, Purkinje cell layer region; RHP, retrohippocampal region; RSP, retrosplenial area; TRN, tegmental reticular nucleus; SPV, spinal nucleus of the trigeminal; and VNC, vestibular nuclei. Scale bar: 500 μm. 146 brain research 1632 (2016) 141–155