Proc. Natl. Acad. Sci. USAVol. 93, pp. 11173-11178, October 1996Neurobiology

Targeted disruption of the cyclin-dependent kinase 5 gene resultsin abnormal corticogenesis, neuronal pathology andperinatal deathToSHIO OHSHIMA*t, JERROLD M. WARD*, CHANG-Goo HUHt, GLENN LONGENECKER*, VEERANNA§,HARISH C. PANT§, RoscOE 0. BRADYt, LEE J. MARTINS, AND ASHOK B. KULKARNI*II*Gene Targeting Research and Core Facility, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892; tDevelopmental andMetabolic Neurology Branch, and §Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health,Bethesda, MD 20892; *Veterinary and Tumor Pathology Section, Office of Laboratory Animal Science, National Cancer Institute, National Institutes of Health,Frederick, MD 21702; and 1Department of Pathology, Neuropathology Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Roscoe 0. Brady, July 3, 1996

ABSTRACT Although cyclin-dependent kinase 5 (Cdk5)is closely related to other cyclin-dependent kinases, its kinaseactivity is detected only in the postmitotic neurons. Cdk5expression and kinase activity are correlated with the extentof differentiation of neuronal cells in developing brain. CdkSpurified from nervous tissue phosphorylates neuronal cy-toskeletal proteins including neurofilament proteins and mi-crotubule-associated protein tau in vitro. These findings in-dicate that Cdk5 may have unique functions in neuronal cells,especially in the regulation of phosphorylation of cytoskeletalmolecules. We report here generation of CdkS(-/-) micethrough gene targeting and their phenotypic analysis.CdkS(-/-) mice exhibit unique lesions in the central nervoussystem associated with perinatal mortality. The brains ofCdkS(-/-) mice lack cortical laminar structure and cere-bellar foliation. In addition, the large neurons in the brainstem and in the spinal cord show chromatolytic changes withaccumulation of neurofilament immunoreactivity. These find-ings indicate that Cdk5 is an important molecule for braindevelopment and neuronal differentiation and also suggestthat Cdk5 may play critical roles in neuronal cytoskeletonstructure and organization.

The cell division cycle in all eukaryotes is controlled by a largefamily of protein kinases, cyclin-dependent kinases, and theirregulatory subunits, cyclins (1, 2). Cyclin-dependent kinase 5(Cdk5) was originally isolated through its sequence homologyto human cdc2 (2-4). However, CdkS expression was foundpredominantly in the brain, and Cdk5 activity, detected only inthe nervous tissue, correlated well with the extent of differ-entiation of neuronal cells in developing brain (5). Recently, aneuron-specific regulatory subunit for Cdk5, p35, has beenidentified and shown to be different from cyclins (6-8).

Neurofilaments (NFs) are composed of triplet proteins oflow (NF-L), medium (NF-M), and high (NF-H) molecularmass. NF proteins are the most extensively phosphorylatedproteins in the adult nervous system in vivo (9, 10), and thephosphorylation of NF proteins is developmentally and spa-tially regulated. Most of this phosphorylation takes place in theLys-Ser-Pro (KSP) repeats in the carboxyl-terminal tail do-main of NF-M and NF-H. This phosphorylation is believed tostabilize the NF network in the axon and to affect the axonaltransport and conduction velocity in the neurons (11). SomeKSP sites in the tail domain of NF-H have been suggested tobe phosphorylated by Cdk5 isolated from rat spinal cord andbovine brain (3, 12). Cdk5/p35 also phosphorylates tau pro-tein, and this phosphorylation occurs exclusively at the samesites found in the tau protein from Alzheimer's disease brain

(13). Moreover, expression of dominant-negative mutants ofCdk5 in neuronal culture inhibits neurite outgrowth (14). Allof these findings indicate that Cdk5 may have unique functionsin neuronal cells, especially in the regulation of phosphoryla-tion of neuronal cytoskeletal proteins.To delineate precise roles of Cdk5 in vivo, we have generated

mice deficient in CdkS expression by gene targeting.CdkS(-/-) mice exhibit perinatal mortality associated withabnormal corticogenesis and cerebellar defoliation. Addition-ally, these mice show chromatolytic changes in large neuronsof the brain and spinal cord. Immunostaining of the NFproteins suggested defective transport of NFs in the affectedneurons.

MATERIALS AND METHODSConstruction of Targeting Vector. The targeting construct

contains 7.3 kb of 129/SvJ-derived Cdk5 genomic sequence(15) in the pPNT vector (ref. 16; see Fig. 1A). The 0.8 kb of5'-flanking fragment, consisting of an NotI-BglII fragmentisolated from lambda 11 clone (15) was subcloned into XhoIsite of pPNT. The 6.5 kb of EcoRI-BglII fragment wassubcloned between EcoRI and BamHI sites of pPNT.Gene Targeting and Generation of Mouse Mutant. Tissue

culture of Jl embryonic stem (ES) cells (17) and conditions forelectroporation of the targeting construct were performed asdescribed (18). Selection with G418 at 350 ,ug/ml and 2 ,Mganciclovir was initiated 24 h after electroporation. After 8-10days, resistant clones were picked, expanded, and analyzed asdescribed (19). Genomic DNA from these clones was digestedwith EcoRI and hybridized with probe A isolated from the0.5-kb Spel-NotI fragment from the 5'-flanking sequence (seeFig. 1A). The sizes of the EcoRI fragments from wild-typeallele and mutated allele were 4.3 kb and 5.5 kb, respectively.The presence of a single integration event was demonstratedby hybridization with neomycin phosphotransferase gene-specific probe (neo probe) using the same membrane afterstriping probe A. Blastocyst injections were performed asdescribed (20). Mice heterozygous for the Cdk5 gene deletion,CdkS(+/-), were generated by mating male chimeras withC57BL/6 females. CdkS(-/-) mutant mice were generatedfrom intermatings of CdkS(+/-) mice. To determine thegenotypes of offspring, tail DNA was digested with EcoRI andhybridized with probe A as described above.

Abbreviations: Cdk5, cyclin-dependent kinase 5; NF, neurofilament;NF-L, -M, and -H, NF of low, medium, and high molecular mass,respectively; ES, embryonic stem; dpc, day(s) postcoitus; dpp, day(s)postpartum."To whom reprint requests should be addressed at: Building 30, Room132, National Institute of Dental Research, National Institutes ofHealth, 30 Convent Drive, Bethesda, MD 20892-4326. e-mail:akulkarni@yoda.nidr.nih.gov.

11173

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11174 Neurobiology: Ohshima et al.

Histology and Immunohistochemistry. For histologicalanalysis, embryos [Cdk5(+/+), n = 8; Cdk5(-/-), n = 8] or

neonatal pups [Cdk5(+/+), n = 12; Cdk5(-/-), n = 101 wereimmersion fixed for 3-5 days either in 10% phosphate-buffered formalin or Bouin's solution. Brain and spinal cordsamples were embedded in paraffin, and tissue sections werecut at 6 ,um and stained with hematoxylin and eosin, Nissl, andBielschowsky's silver. For immunohistochemistry, Bouin-fixedparaffin-embedded sections were used for the avidin-biotin-peroxidase complex (ABC) technique (Vector Laboratories)with diaminobenzidine. Monoclonal antibodies against NFs(SMI-31 at 1:10,000 and SMI-32 at 1:1000; Sternberger Mono-clonals, Baltimore) were used as primary antibodies (21).RNA Isolation and Northern Blot Analysis. RNA was

isolated from individual brains at 16.5 days postcoitus (dpc) bythe acid guanidium thiocyanate-phenol-chloroform method(22). Twenty micrograms of total RNA from brain was heat-denatured and size-fractionated by electrophoresis through a

1.0% formaldehyde/agarose gel and transferred onto a nylonmembrane (Nytran; Schleicher & Schuell) by capillary blottingas described (23). Hybridization was performed using themouse Cdk5 cDNA generated by reverse transcriptase-PCR(14) in 6x standard saline citrate (SSC)/50% formamide at42°C overnight. The filter was washed twice in 0.1 x SSC/0.1%SDS at 50°C for 30 min. After stripping the probe, the same

filter was used for hybridization with the mouse GAPDHprobe (19).Western Blot and Kinase Assay of Cdk5 Kinase Activity.

Western blot analysis for Cdk5 protein was performed usingCdk5 monoclonal antibody (Oncor) as a primary antibody.Cdk5 kinase activity was measured using an analog peptide forNF-H tail KSPXK repeats, VKSPAKEKAKSPEK, as a sub-strate (12, 24).

RESULTS

Targeting of the CdkS Gene. Genomic clones of the mouse

Cdk5 gene were isolated from the 129/SvJ mouse genomiclibrary, and the gene structure was characterized as described(15). These genomic fragments were used to construct thetargeting vector, which carried a 0.8-kb deletion including a

part of exon III, and exons IV and V. This deletion wasreplaced by a neomycin resistance gene as a positive selectionmarker. Negative selection against random integration wasconferred on the vector by a herpes simplex virus thymidinekinase (tk) gene (25).

Ji ES cells were electroporated with the targeting vectorDNA, and, subsequently, 238 resistant clones were isolated andscreened by Southern blot hybridization analysis (Fig. 1B).This revealed five targeted clones positive for the predictedsize of the targeted allele. Additional integration of thetargeting vector was excluded by screening these clones byhybridizing the same membrane with the neo probe (Fig. 1B).Selected clones were injected into blastocysts to generatechimeric mice. Out of six male and one female chimeras with>90% coat color chimerism, five male chimeras successfullytransmitted Cdk5 mutation through the germ line.

Northern blot analysis was performed to confirm completeinactivation of the Cdk5 gene. Total RNA from 16.5 dpcembryos was hybridized with Cdk5 cDNA probe. Cdk5 mRNAcould not be detected in Cdk5(-/-) embryos (Fig. 1D).Furthermore, Western blot analysis revealed 31.5-kDa Cdk5protein in wild-type brain, but not in Cdk5(-/-) brain (Fig.1E). Finally, the complete inactivation of Cdk5 activity was

confirmed by a kinase assay using an analog peptide for NF-Htail KSPXK repeats (Fig. 1F).

Early Lethality of Cdk5(-/-) Mice. Cdk5(+/-) miceappeared normal. To assess the homozygous mutant pheno-type, Cdk5(+/-) mice were interbred, and at 10 days post-partum (dpp) were genotyped (Table 1). Of 55 progeny, 18

were CdkS(+/+), 37 were Cdk5(+/-), and none wereCdk5(-/-), indicating early lethality of Cdk5(-/-) mice.To determine the timing of Cdk5(-/-) mice lethality,

newborns and embryos of different ages were genotyped(Table 1). At 16.5 dpc, CdkS(-/-) embryos were found inexpected percentage (24%), but at 18.5 dpc and at 0 dpp, thepercentage of CdkS(-/-) mice was found to be reduced to19% and 9%, respectively. These data indicate that 64% ofCdkS( - / -) mice die in utero after 16.5 dpc. NewbornCdk5(-/-) mice were found either dead or weak with hy-popnea, reduced mobility of four limbs and cyanosis leading tomoribund condition and death within 12 h after birth. Appar-ently healthy CdkS(- I-) newborns, shortly after birth, failedto respond to clamp stimulation of their tails. All of theCdk5(- /-) newborns analyzed for gross pathology revealed alack of milk in their stomach.CdkS(-/-) Mice Exhibit Abnormal Corticogenesis and

Unique Neuronal Pathology. The dominant expression ofCdk5 in the nervous tissue (2-4) and the limited expression ofits kinase activity in nervous tissue (5) were suggestive ofpossible neuronal dysfunction in CdkS(-I-) mice. His-topathological analysis of 18.5 dpc embryos and newbornCdk5(- /-) mice revealed lesions only in the brains and spinalcords, but not in any other tissues including heart, lung, liver,and kidney.Normal stratification of neocortical neurons in the cerebral

cortex was absent in Cdk5(-/-) mice (Fig. 2 B and D).Abnormal stratification was also seen in the hippocampalformation where pyramidal neurons were not organized dis-cretely (Fig. 2 B and F). The cerebella of Cdk5(-/-) micelacked the typical foliation and tripartite layering (Fig. 2H).The large neurons in the cranial nerve nuclei of the brain stemof CdkS(-I-) mice displayed ballooned perikarya with aneccentrically positioned nucleus and dispersed Nissl substancesuggestive of chromatolytic changes (Fig. 2J). In the facialnerve and hypoglossal nerve nuclei, motor neurons alsoshowed cytoplasmic vacuoles (Fig. 2L). These abnormalitieswere also seen in the motor neurons of the ventral horn of thespinal cord in Cdk5(- /-) mice (Fig. 2N). On the other hand,the dorsal root ganglia remained normal in appearance inCdkS(-/-) mice, indicating the restriction of these degener-ative changes to the central nervous system.Accumulation of NF Protein Immunoreactivity in the Neu-

rons of CdkS(-/-) Mice Brain. Bielschowsky's silver stainingrevealed significant positive staining of axonal structures andfew neurons in the wild-type mouse brain. Numerous positivelystained large neurons were seen in the brain stem of theCdk5(- /-) mice (data not shown) indicating possible accu-mulation of phosphorylated NFs in the affected neurons. Weperformed immunostaining of the brain sections fromCdk5(+/+) and CdkS(-/-) mice at 18.5 dpcwith monoclonalantibody against phosphorylated NFs, SMI-31 (21). In brainstems of CdkSx(+/+) mice, anti-phosphorylated NFs antibodystained many axons and few neuronal cell bodies (Fig. 3A). Incontrast, numerous large neuronal cell bodies of CdkS(- /-)brains were stained positive with anti-phosphorylated NFsantibody (Fig. 3 B and C). Most of the positive neuronal cellbodies in the brain stem of CdkS(- /-) mice were also stainedwith anti-nonphosphorylated NFs antibody, SMI-32 (Fig. 3D).Accumulationi ofNF protein immunoreactivity in the neuronalcell bodies suggest defective transport of NFs.

DISCUSSIONWe have generated Cdk5(- / -) mouse lines by gene targeting.Neither Cdk5 mRNA nor Cdk5 protein (and its kinase activity)were detected in these mice, indicating a successful disruptionof the Cdk5 gene. More than 60% of CdkS(-/-) mice die inutero, and the newborns become weak and die within 12 h afterbirth. Histopathological analysis revealed abnormal cortico-

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 11175

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genesis and unique neuronal lesions with accumulation of NFprotein immunoreactivity, suggestive of defective axonal trans-port of NFs. This phenotype demonstrates a critical role for

Table 1. Genotypes of litters from Cdk5(+/-) intercrosses

No. of GenotypeAge pups +/+ +/- -- (% of KO)

12.5 dpc 17 4 9 4 (23.5%)16.5 dpc 76 20 38 18 (23.6%)18.5 dpc 138 36 76 26 (18.8%)0 dpp 339 105 203 31* (9.1%)10 dpp 55 18 37 0* (0.0%)Neonates and embryos were harvested at the times indicated.

Genomic DNA was extracted from the tail of each pup, and subjectedto Southern blot analysis to determine the genotype.*A x2 goodness of fit test was performed at each stage for genotyperatio; at 0 ddp and 10 dpp, CdkS(-/-) genotype was significantlyreduced (P < 0.005).

Cdk5 in corticogenesis and in the normal cytoskelatal archi-tecture of the developing neurons.

Abnormalities of neuronal migration in the developing brainmay result in the lack of normal stratification in the brain andabsence of foliation in cerebellar cortex of CdkS(-/-) mice.Recently, the association of Cdk5 activator subunit witha-actinin was reported using the yeast two-hybrid system (14).a-Actinin can function as a cytoskeletal mechanotransducerfrom the cell surface in association with cell movement (26).A critical role of Cdk5 in neurite outgrowth was suggestedusing a dominant-negative mutant system in neuronal culture(14). Thus, one possible explanation of neuronal pathologyseen in Cdk5(-/-) mice is perturbations in neurite outgrowthand axonal pathfinding. The chromatolytic-like changes inlarge neurons in hindbrain of CdkS(-/-) mice are suggestiveof neuronal response to axonal injury (27). Degenerativechanges in large neurons of the brain stem, including motorneurons in the lower cranial nerve nuclei and spinal cord, maybe the cause of the early lethality observed in CdkS(-/-)mice.

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FIG. 2. Neuropathological analysis of CdkS(+/+) (A, C, E, G, I, K, and M) and CdkS(-/-) mice (B, D, F, H, J, L, and N). Nissl-stained sagittalsections showing neocortex and underlying hippocampus (A and B) at low magnification (x75), as well as neocortex (C and D) and hippocampus(E and F) at higher magnification (x 150). Hematoxylin and eosin stained cerebellum in sagittal section (G and H; X75). CdkS(-/-) brain lackedcortical lamination of neurons (B and D), and hippocampus failed to be organized into a typical three-layered structure (B and F). CdkS(- /-)cerebellum lacked foliation and failed to segregate into layers (H). Hematoxylin and eosin stained coronal sections of the medulla (I and J), andNissl stained coronal section of facial nerve nuclei (K and L; X300) and transverse sections of ventral horn motor neurons of the lumbar spinalcord (M and N; X300). The large neurons had ballooned cell bodies with dispersed Nissl substance and eccentric nuclei (see arrows in J, L, andN). In addition to these changes, motor neurons in the facial nerve nuclei and the ventral horn of the spinal cord displayed vacuolations (see arrowsinL and N).

A number of neuron-specific proteins, including NF proteins motif. Cdk5/p35 has been suggested to phosphorylate neuro-(28, 29), synapsin (30), and microtubule-associated protein tau nal cytoskeletal proteins including NF-H and NF-M (4, 12),(31), are phosphorylated in vivo on the p34Cdc2 consensus tau protein (13), and MAP2 (14) in vitro. Lysine-rich histone

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 11177

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FIG. 3. Immunohistochemical analysis of NFs in CdkS(+/+) (A)and Cdk5(-/-) (B-D) brains. Brain stem in sagittal section immu-nostained with anti-phosphorylated NFs antibody (A-C) and withanti-nonphosphorylated NFs antibody (D) (20). Numerous phosphor-ylated NF-positive neurons were seen in the brain stem (B and C) ofCdk5(-/-) mice, while the normal axonal localization of phosphor-ylated NFs was seen in Cdk5(+/+) brain stem (A). Most of thephosphorylated NFs-positive neuronal cell bodies were also stainedwith anti-nonphosphorylated NFs antibody (D).

(H1) can also be efficiently phosphorylated by Cdk5 purifiedfrom nervous tissue (4). Phosphorylation of these neuronalcytoskeletal proteins are developmentally regulated and con-sidered to have significant roles on neuronal differentiationand functions.

Phosphorylation is believed to modulate axonal transport ofNFs (32). Depending on their phosphorylation states, NFsshow different localizations in the neurons: the phosphory-lated forms are found in the axons, while the nonphosphory-lated forms are found in neuronal cell bodies and in dendrites(21, 33). The axonal NFs are phosphorylated posttranslation-ally, as newly synthesized NF proteins in cell body are trans-ported and incorporated in the axonal cytoskeleton (34-36).Cdk5-mediated phosphorylation of the specific motif, KSPXK,present in the tail domain of NF-H and NF-M, results in thechange of NF conformation (12, 37, 38), as well as in theirassociation with microtubules (37, 38). Impairment of NFtransport is, thus, another possible cause of neuronal pathol-ogy seen in Cdk5(- /-) mice. The neurofibrillary pathology oraccumulation of NF protein immunoreactivity in perikarya oflarge neurons in brain stem and spinal cord of Cdk5(- / -)

mice is reminiscent of complex cytoskeletal pathology seen inseveral neurodegenerative disorders, such as motor neurondisease (39), progressive supranuclear palsy (40), Lewy bodydisease (41), and chemical intoxication by neurotoxic sub-stances such as aluminum (42, 43), 3,/-iminodiproprionitrile(IDPN; ref. 44), and 2,5-hexanedione (45). These similaritiesimplicate a pathophysiological link between lack of Cdk5function in Cdk5(-/-) mice and human neurodegenerativedisorders. The higher immunoreactivity of the phosphorylatedNFs seen in large neuronal perikarya of CdkS(- /-) mice alsosuggests the presence of other NF kinases and their possiblecompensatory effects.

In conclusion, CdkS(-/-) mice exhibit a distinct phenotypeand provide a valuable animal model to study the precise rolesof Cdk5 not only in developing mouse brain but also inpathological processes underlying some of the human neuro-degenerative disorders. A detailed analysis of structural orga-nization of the cytoskeletal proteins in Cdk5(-/-) neurons isunderway. These studies should provide insight into the neu-rocytoskeletal pathology in numerous disorders of the nervoussystem. Most importantly, this phenotype warrants a carefulexamination of Cdk5 functions in the mature and agingnervous system, which is often susceptible to debilitatingdisorders.

We thank M. ladarola, H. K. Kleinman, K. M. Yamada, and Y.Yamada for critical reading of the manuscript; K. Suzuki for helpfulcomments on pathological findings; R. Mulligan for the pPNT vector;and J. Drago and H. Westphal for Jl cells.

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