INTRODUCTIONNeuronal polarization, involving the initiation of immature neurites,the specification and formation of axon and dendrites, and theformation of synaptic contacts, is essential for signal transmissionin the central nervous system (CNS) (Arimura and Kaibuchi, 2007;Dotti et al., 1988). Proper microtubule organization is crucial forthis process (Lefcort and Bentley, 1989; Hirokawa and Takemura,2005; Kimura et al., 2005; de Anda et al., 2005). Thus, disruptionof microtubule structure, or of the formation of the myelin sheaththat surrounds the axons, are associated with several pathologies,including axon degeneration (Coleman and Perry, 2002; Perrin etal., 2005) and rare myelin protein mutations (e.g. Pelizaeus-Merzbacher disease) (Garbern, 2005; Zhao et al., 2001). Axondegeneration is also found in various neurodegenerative disorderssuch as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),Alzheimer’s disease (AD), Parkinson’s disease (PD) and others(Coleman and Perry, 2002; Perrin et al., 2005).

Liver kinase B1 (LKB1; also called STK11) is a protein kinaseand potent tumour suppressor. Mutations in the LKB1 gene resultin Peutz-Jeghers syndrome (PJS) (Boudeau et al., 2003). LKB1 is apartial mammalian homologue of three kinases in Saccharomycescerevisiae, Elm1, Pak1 and Tos3; these kinases phosphorylate SNF1,the yeast homologue of mammalian AMP-activated protein kinase(AMPK) (Sutherland et al., 2003). LKB1 is one of three upstreamkinases for AMPK in mammalian cells (Hawley et al., 2003; Woods

et al., 2003) and also phosphorylates 12 further kinases in theAMPK subfamily (Lizcano et al., 2004).

LKB1 is implicated in the control of cell polarity, and homologuesof LKB1 in Drosophila melanogaster (dLKB1) and Caenorhabditiselegans (PAR-4) control epithelial cell polarity (Jansen et al., 2009).AMPK has been proposed as a further probable mediator of theeffects of LKB1 on cell polarity (Zhang et al., 2006). In neurons,LKB1 is thought to control neuron polarity by affecting axondifferentiation (Shelly et al., 2007; Barnes et al., 2007). The lattereffects are thought to be mediated via the inactivation of a signaltransduction cascade that activates late-onset sporadic Alzheimer’sdisease (SAD)-A/B kinases, also called Brsk1/2 (Barnes et al., 2007).In C. elegans, the homologue of the LKB1 binding partner STRADa,termed STRD-1, binds together with LKB1 to form a tightlyassociated functional complex with the C. elegans SAD-A/B kinaseSAD-1 to organize synaptic proteins and establish neuron polarity(Kim et al., 2010). SAD-A/B kinase is thought to act byphosphorylating tau, a microtubule stabilization protein (Kishi etal., 2005), at Ser262.

Despite the above evidence, a direct demonstration that LKB1is involved in these processes in vivo in adult mammals is lacking.Cre expression under rat insulin 2 promoter (RIP2) has been foundin mid and ventral brain and spinal cord. Using mice lacking LKB1in these regions (Wicksteed et al., 2010), we demonstrate here thatLKB1 in certain brain areas is required for axon stability and normalhind-limb locomotor control.

RESULTSMice lacking LKB1 owing to the RIP2-Cre transgene develop hind-limb paralysisTo generate mice lacking LKB1 in the mid and ventral brain, andin the spinal cord from embryonic day 11.5 (E11.5) (Gannon et al.,2000; Wicksteed et al., 2010), we crossed mice bearing floxed LKB1alleles with RIP2-Cre mice (Sun et al., 2010b). We have previouslyshown that these mice (LKB1KO) are hyperinsulinemic andmildly hypophagic due respectively to deletion of LKB1 in the

Disease Models & Mechanisms 193

Disease Models & Mechanisms 4, 193-202 (2011) doi:10.1242/dmm.006833

1Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism,Department of Medicine, and 2Wolfson Neuroscience Laboratories, Faculty ofMedicine, Imperial College London, London, SW7 2AZ, UK*Author for correspondence (g.rutter@imperial.ac.uk)

Received 13 September 2010; Accepted 28 October 2010

© 2011. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms

SUMMARY

Axon degeneration is observed in neurodegenerative diseases and neuroinflammatory disorders, such as Alzheimer’s disease, Parkinson’s diseaseand multiple sclerosis. The molecular basis of this process remains largely unknown. Here, we show that mice deleted for the tumour suppressorLKB1 (also called STK11) in the spinal cord, some parts of the brain and in the endocrine pancreas (LKB1KO mice) develop hind-limb dysfunctionand axon degeneration at about 7 weeks. Demyelination and macrophage infiltration are observed in the white matter of these mice, predominantlyin the bilateral and anterior funiculi of the thoracic segment of the spinal cord, suggesting damage to the ascending sensory signalling pathwayowing to LKB1 deletion in the brain. Microtubule structures were also affected in the degenerated foci, with diminished neurofilament and tubulinexpression. Deletion of both PRKAA1 genes, whose products AMPKa1 and AMPKa2 are also downstream targets of LKB1, with the same strategywas without effect. We thus define LKB1 as an intrinsic suppressor of axon degeneration and a possible target for strategies that can reverse thisprocess.

RIP2-mediated LKB1 deletion causes axon degenerationin the spinal cord and hind-limb paralysisGao Sun1, Richard Reynolds2, Isabelle Leclerc1 and Guy A. Rutter1,*

RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

pancreatic -cell and in a small population of hypothalamic neurons(Sun et al., 2010b). In addition, as reported below, LKB1KO micedeveloped dysfunction of both hind limbs at approximately 7 andeight 8 for females and males, respectively. This was characterizedby clumsy and autonomous twitching of both hind limbs (Fig.1A,B). At 1-2 weeks after the initial onset of an observableabnormality, both legs of LKB1KO mice became paralyzed.However, as assessed by toe pinching, basal muscle reflexes in thehind limbs of LKB1KO mice still existed at this time point, whichis indicative of unaffected muscle function. Moreover, both front

limbs of LKB1KO mice maintained mobility (Fig. 1B), and themice were still able to gain access to food and water 2 weeks afterthe initial onset of symptoms. The latter findings suggested thatthe upper section (cervical) of the spinal cord was less affected thanthe lower section by LKB1 deletion. Furthermore, before paralysisonset, LKB1KO mice showed similar mobility to wild-type controlmice. However, after the onset of hind-limb dysfunction, LKB1KOmice displayed significantly reduced mobility as characterized byan unwillingness to move and by crouching, both of which areindications of possible trauma. In addition to the above changes,

dmm.biologists.org194

LKB1 and axon degenerationRESEARCH ARTICLE

Fig. 1. LKB1KO mice develop hind-limb paralysis.(A)Representative image and (B) foot-print of a LKB1KOmouse 1 week after the initial onset of hind-limbdysfunction, compared with a wild-type control. Scale bar:10 mm. (C)Hind-limb dysfunction onset curve and (D)survival curve for male and female LKB1KO mice and theirwild-type controls. 100% indicates a completely healthystatus, whereas 0% indicates onset of hind-limb dysfunction(clumsy and autonomous twitch of hind-limbs) and death.(E)Weight increases of LKB1KO mice and wild-typecontrols 1 and 2 weeks after the initial onset of hind-limbdysfunction. Data are expressed as means ± s.e.m.; *P<0.05,***P<0.001. n10-12 mice per genotype.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

LKB1KO mice displayed complete loss of control of their tails 1-2 weeks after the initial onset of the hind-limb dysfunction (Fig.1A). A large reduction in body weight increases was also observedup to 2 weeks after paralysis, after which point LKB1KO micedisplayed rapid weight loss and loss of urinary control (data notshown). In all cases, the animals died 4-5 weeks after the initialonset of paralysis (Fig. 1C).

Recent studies on RIP2-Cre expression in adult mice byWicksteed and his colleagues showed extensive expression of Crein the mid and ventral brain (Wicksteed et al., 2010). In the presentstudy, Cre and LKB1 expression were also studied in the spinalcord: analysis of LKB1 expression in the cervical, thoracic andlumbar regions of the spinal cord of LKB1KO mice byquantitative reverse transcription PCR (qRT-PCR) using primersflanking exon 3 and 6, and western (immuno)blotting analysisusing anti-LKB1 antibody raised against the C-terminal of LKB1,demonstrated that RIP2-Cre-mediated recombination occurredsubstantially in the thoracic area, and to a lesser extent in thecervical and lumber areas (supplementary material Fig. S1A,B).Correspondingly, immunohistochemical analysis of the spinalcord from LKB1KO mice revealed lowered numbers of cells thatwere intensely stained for LKB1 in the grey matter of thoracicregions (supplementary material Fig. S1C) compared with wild-type controls.

LKB1KO mice develop axon degeneration in the white matter ofthe thoracic segment of the spinal cordTo try to understand the molecular and cellular basis of the abovechanges, post-mortem analysis of different organs extracted fromLKB1KO mice was performed 1 or 7 days after the initial onset ofhind-limb dysfunction. The tissues analyzed included: whole brain(cerebral cortex, hypothalamus and cerebellum), heart, lung, liver,spleen, kidney, skeletal muscle of hind limb, pelvis, sciatic nerve andspinal cord. Haematoxylin and eosin (H&E) staining was used forgeneral morphological analysis and luxol fast blue (LFB) was usedto detect myelin. This analysis revealed several large foci of axondegeneration in the white matter of the thoracic region of LKB1KOmouse spinal cord 7 days after the initial onset of hind-limb disability(Fig. 2Ad,Ai), whereas fewer and smaller foci only were found 1 dayafter initial onset (Fig. 2Ac,Ah). In the area where degenerationoccurred, axon disintegration and demyelination were evident byusing LFB staining, with a loss of dark blue myelin staining and theformation of large digestion chambers caused by myelin vacuolation(Fig. 2Af,Ah,Ai,Bb,Bd,Bf,Bh). Notably, brightly eosinophilic acellularspheroids were present in some of the vacuolated chambers whenthe sections were stained with H&E (Fig. 2Ac,Bd, arrows), suggestingpossible necrosis or swollen axons (Fig. 2Bd,Bh) (Fujimura et al.,2009). At 7 days after the onset of hind-limb dysfunction in LKB1KOmice, a complete loss of myelin staining and the disappearance ofaxons was observed in the thoracic region of the spinal cord (Fig.2Ad,Ai,C,D). A striking increase in the overall number of cell nucleiwas also evident at the foci of degeneration of LKB1KO mice 1 dayafter the initial onset of hind-limb dysfunction, which is suggestiveof inflammation and macrophage infiltration, and this increasecontinued as degeneration progressed (Fig. 2Ac,Ad,Ah,Ai and Fig.4A). In addition to the large foci of axon degeneration, several smallerfoci with fewer and smaller digestion chambers and vacuolated axonswere found in the thoracic area of the spinal cord (Fig. 2Ae,Aj). In

these areas, axons were not completely disintegrated, but stillretained a partial myelin sheath, which was detached from the axons.

LKB1KO mice display impaired sensory signalling pathways inthe thoracic segment of the spinal cordTransverse sections of this area in LKB1KO mouse spinal cord 1day after the initial onset of hind-limb dysfunction revealed thedegeneration foci mainly in the lateral and anterior funiculi, includingin the spinothalamic and spinocerebellar tracts of the spinal cord,indicating damage primarily to the ascending sensory signallingpathways (Fig. 2Ba,Bc,Be,Bg). By contrast, changes to the descendingmotor systems were less evident, suggesting that alterations in thesepathways, which are likely to underlie the hind-limb paralysis, mightoccur either in higher brain regions in which RIP2-Cre-mediateddeletion is also likely to occur, or were sufficiently diffuse in thethoracic region of the spinal cord to escape from histologicaldetection. Examination of LKB1KO mice before paralysis alsorevealed several sites of mild demyelination in the thoracic regionof the spinal cord (Fig. 2Ab,Ag,C,D), a phenotype similar to that seenin the cervical and lumbar areas of the spinal cord in LKB1KO mice7 days after the onset of symptoms (Fig. 2C,D; supplementarymaterial Fig. S2).

A limited degree of LKB1 deletion was previously found in theventromedial hypothalamus of LKB1KO mice (Sun et al., 2010b),consistent with earlier studies using RIP2-Cre deleter mice bredwith a lacZ reporter strain (Gannon et al., 2000; Choudhury etal., 2005; Lin et al., 2004). However, close examination of thehypothalamus and other parts of the brain, including theforebrain, cortex and cerebellum, did not reveal any evidence forthe presence of degenerating axons in these regions of LKB1KOmice before or after hind-limb paralysis (supplementary materialFig. S3).

LKB1KO mice display disintegration of neurofilaments andmicrotubules in the spinal cordTo examine the effects of LKB1 deletion on the formation ofmicrotubules and neurofilaments within spinal cord axons, thedegenerated areas were stained with markers for neurofilaments[neuron filament 165kD (NF165kD; 2H3); Fig. 3A], tau (Fig. 3A) and-tubulin III (data not shown). Affected sections of LKB1KO mousespinal cord displayed diminished NF165kD, tau and -tubulin IIIstaining in an age-dependent manner. Thus, disorganization,disintegration and partial loss of neurofilaments and microtubulesin the foci of spinal cord were apparent in mice 1 day after the initialonset of hind-limb dysfunction, and almost total loss of signals wasevident 1 week from the initial onset, indicating severe axondegeneration (Fig. 3A). Several neurofilament-containing spheroids(arrows in Fig. 3A) were also evident in the degenerated areas. Thesefindings were confirmed by western (immuno)blotting analysis ofprotein extracts from thoracic regions of the spinal cord fromLKB1KO mice. Reduced NF165kD and tau immunoreactivity wasalso evident in spinal cord sections from LKB1KO mice 1 day afterthe initial onset of paralysis (Fig. 3B).

LKB1KO mice develop increased macrophage invasion after theinitial onset of hind-limb dysfunctionMacrophage invasion is commonly observed during axondegeneration (Zhang and Guth, 1997; Buss et al., 2004). In view of

Disease Models & Mechanisms 195

LKB1 and axon degeneration RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

the increase in apparent cell number (Fig. 4A), but diminishednumber of axons (Fig. 2C), in foci in the spinal cord of LKB1KOmice, we next explored the possibility that inflammatorymacrophage infiltration might be involved in the present model.Consistent with this view, progressive macrophage invasion in areasof axon degeneration was revealed by periodic acid Schiff (PAS)staining for glycogen and other carbohydrate-containingmacromolecules in active macrophages (Fig. 4B) (Sobolev, 1959).This was further confirmed by F4/80 immunofluorescence stainingfor these cells (Leenen et al., 1994) (Fig. 4C). However, neither thethoracic areas of the spinal cord of LKB1KO mice before the onsetof hind-limb dysfunction, nor the cervical or lumbar segments fromthese mice after paralysis, showed any positivity for PAS or F4/80staining (data not shown).

Deletion of AMPKa1 and AMPKa2 catalytic subunits does notaffect spinal cord morphology or motor functionGiven the role of AMPK in mediating the effects of LKB1 in thecontrol of cell polarity in other systems (see the Introduction),we further investigated the role of this enzyme in mediating thespinal cord degeneration observed in LKB1KO mice. In contrastto animals deleted for LKB1 in the spinal cord and other regionsusing the RIP2-Cre deleter strain, mice generated using the sametransgene to delete both AMPKa1 and AMPKa2 subunits in thesame cell types (AMPKdKO mice) (Sun et al., 2010a) did notdisplay any locomotor abnormalities (Fig. 5A) nor altered survivalcurves (not shown) compared with heterozygous or wild-typeanimals. Moreover, close examination of the thoracic and othersegments of the spinal cord from AMPKdKO mice did not reveal

dmm.biologists.org196

LKB1 and axon degenerationRESEARCH ARTICLE

Fig. 2. Histological changes inwhite matter in the thoracicregion of spinal cord of LKB1KOmice. (A)Representative H&Estaining (a-e) and LFB staining (f-j) ofthe white matter of the thoracicregion of spinal cord from wild-typemice (a,f ) and LKB1KO mice before(b,g), or 1 day (c,h) or 7 days (d,i)after the initial onset of hind-limbdysfunction, or of the less-affectedwhite matter of the thoracic regionof spinal cord from LKB1KO mice 1day after the initial onset of hind-limb dysfunction (e,j). Arrowsindicate eosinophilic spheroid. Scalebar: 50m. (B)Representative H&Estaining (a-d) and LFB staining (e-h)of transverse sections of the thoracicregion of spinal cord from wild-typemice (a,b,e,f ) and LKB1KO mice 1day after initial onset of hind-limbdysfunction (c,d,g,h). Arrows indicateeosinophilic spheroid. Scale bars:100m (a,c,e,g); 50m (b,d,f,h).(C,D)Quantification of degenerating(C) and demyelinated (D) axons inthoracic, cervical and lumbar regionsof white matter spinal cord fromLKB1KO mice before or after 1 dayand 7 days of initial onset of hind-limb dysfunction, and their wild-typecontrols. Intensity of axon stainingperm2 area of white matter weremeasured based on NF165kD (C)and LFB staining (D) using ImageJ.*P<0.05, **P<0.01, ***P<0.001. n4mice per genotype.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

any changes in axon or myelin formation (Fig. 5B). Thus, LKB1seems to control axon stability in the spinal cord independentlyof AMPK.

DISCUSSIONRIP2-Cre-mediated LKB1 deletion leads to hind-limb paralysis byaffecting axon stabilityWe recently noted, in performing a metabolic characterization ofmice deleted for LKB1 in the endocrine pancreas and a restrictedset of CNS neurons using a RIP2-Cre transgene (Sun et al., 2010b),that older animals became paralyzed. The principal aim of thepresent study was thus to dissect the pathology behind this changeand, in doing so, to determine the role of LKB1 in regulatingneuronal polarity and survival in the CNS in vivo.

Although mice null for LKB1 throughout the body die beforeE11.5, the use of an Emx1-Cre deleter strain to allow deletion inpyramidal neuron progenitors demonstrated that LKB1 is requiredfor the polarization of cultured neurons from the neonatalhippocampus and cortex (Barnes et al., 2007; Shelly et al., 2007).We therefore reasoned that LKB1 might play a similar role in axondevelopment and, importantly, in signal transmission along thespinal cord. Given the crucial role of the spinal cord for the normalcontrol of motor function, we further reasoned that deletion ofLKB1 in neurons that extend their axons along the spinal cord andin neurons in the spinal cord might allow us to study the importanceof LKB1 function in neurons in the adult animal.

We therefore generated a mouse model in which LKB1 wasdeleted by RIP2-driven Cre expression. In addition to near completeloss of LKB1 in pancreatic -cells, resulting in a substantial increasein pancreatic -cell mass and hyperinsulinism (Sun et al., 2010b),this approach also led to the elimination of LKB1 from a subset ofneurons within the CNS (see Results and below), including thosein the mid and ventral brain (Wicksteed et al., 2010) and spinalcord (this study). We would stress that it is unlikely that increasedcirculating insulin levels (Sun et al., 2010b) drive the dramaticalterations that we observed in CNS neuron survival within thespinal cord. Thus, similar elevations in circulating insulin occur ina number of other transgenic mouse models as a result of -cellhyperplasia (Remedi et al., 2006; Hennige et al., 2003; Mori et al.,2009b), with no reported effects on CNS function, motor controlor mortality.

The striking finding of this study is that RIP2-Cre-mediated LKB1deletion in a restricted set of neurons leads to severe hind-limbdysfunction and paralysis in young adult mice and eventually topremature death. The white matter of thoracic spinal cord ofLKB1KO mice displayed morphological changes, displaying someof the characteristics of axon degeneration: large foci of disrupted,vacuolized and demyelinated axons with disorganizedmicrotubules. The above changes were accompanied bymacrophage infiltration, a feature that is very similar to that foundin humans (Becerra et al., 1995; Buss et al., 2004) and rats (Zhangand Guth, 1997; Buss and Schwab, 2003) after spinal cord injury,and in P301L tau-expressing transgenic mice, in which themicrotubules of spinal cords were also disrupted (Lewis et al., 2000;Lin et al., 2005). To characterize the neurons in which LKB1 waspossibly deleted, transverse sections of thoracic spinal cord fromLKB1KO mice were closely assessed and the affected focidemonstrated widespread areas of axon degeneration in spinaltracts containing axons involved in predominantly ascendingsensory pathways. Although deletion of LKB1 in LKB1KO micewas also seen in spinal cord, given that most of the neuron cellbodies of the degenerated axons in this location reside in the brain,

Disease Models & Mechanisms 197

LKB1 and axon degeneration RESEARCH ARTICLE

Fig. 3. Disorganization of microtubule structures in axons in the thoracicregion of the spinal cord of LKB1KO mice. (A)Representativeimmunofluorescence staining for NF165kD using mouse anti-NF165kDantibody (1:100; red), and mouse anti-tau antibody (1:50; green) of thethoracic region of wild-type and LKB1KO mice spinal cord 1 day or 7 daysafter the initial onset of hind-limb dysfunction. Arrows indicate neurofilamentcontaining spheroid. W, white matter; G, grey matter. Scale bar: 25m.(B)Western (immuno)blot analysis of Tau and NF165kD levels in LKB1KOmice spinal cord after 1 day of hind-limb dysfunction. GAPDH is used as aloading control.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

we propose that deletion of LKB1 in these neurons in the brainleads to interruption of cell polarity and axon degeneration in thespinal cord, which eventually causes hind-limb paralysis. Notably,examination of neuron cell morphologies in the forebrain cortex,hypothalamus and cerebellum, regions that have been shown byothers to express Cre (Wicksteed et al., 2010), did not reveal anyobvious axon degeneration phenotype. This observation might,however, reflect the expression of Cre in a limited population ofneurons in these areas, such that axon degeneration, if it occurredat all, was difficult to observe. What molecular mechanismunderlies the effects of LKB1 deletion in neurons that form axonsalong the spinal cord? Deletion of LKB1 in pyramidal neurons fromneonatal cortex shows disruption of axon formation, with decreasedSAD-A/B phosphorylation and the phosphorylation of itsdownstream target tau (Barnes et al., 2007). Although it is difficultto monitor phosphorylation levels of SAD-A/B or tau in RIP2-Creneurons owing to the limited distribution of these neurons in thewide area of brain and low expression of Cre in these neurons, the

work of Barnes and his colleagues suggest that an LKB1-SAD-tau(phosphorylated on Ser262) signalling pathway is important incontrolling neuronal polarity in spinal cord. In addition, transgenicmice overexpressing human tau protein containing the FTDP-17mutation (P301L) display behavioural abnormalities with symptomsof Wallerian degeneration in the spinal cord but with increased tau(S262) phosphorylation (Lewis et al., 2000; Lin et al., 2005).Interestingly, Biernat and Mandelkow (Biernat and Mandelkow,1999) reported that hypophosphorylation of tau (S262), resultingfrom mutation of the KXGS domain, inhibited outgrowth ofextensions in Sf9 cells. Conversely, hyperphosphorylation of tau atS262 destabilized microtubules and axons (Biernat et al., 1993;Biernat and Mandelkow, 1999). Finally, Kishi and colleaguesobserved reduced tau (S262) phosphorylation and reduced axonextension in neurons from hippocampus and cortex of SAD-A/B-null mice (Kishi et al., 2005). Thus, the maintenance of anappropriate but limited degree of tau phosphorylation is likely tobe essential for normal neuronal polarization and stability.

dmm.biologists.org198

LKB1 and axon degenerationRESEARCH ARTICLE

Fig. 4. Macrophage infiltration in the thoracicregion of the spinal cord of LKB1KO mice.(A)Nuclei number counts perm2 area of white matterthoracic region of LKB1KO mouse spinal cord beforeor after 1 day and 7 days of initial onset of hind-limbparalysis, and those of their wild-type controls.***P<0.001. n4 mice per genotype. (B)RepresentativePAS staining of white matter of the thoracic region ofthe spinal cord of wild-type and LKB1KO mice after 1day or 7 days of initial onset of hind-limb dysfunction.Note the deep-purple PAS-positive cells in the spinalcord of LKB1KO mice. Scale bar: 50m.(C)Immunofluorescence staining for F4/80, usingrabbit anti-F4/80 antibody (1:100; green), of whitematter of the thoracic region of the spinal cord of wild-type and LKB1KO mice after 7 days of hind-limbdysfunction. Scale bar: 25m.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Nonetheless, direct evidence for this contention would require thesimultaneous inactivation of SAD-A/B in RIP2-Cre neurons, andthus lies outside the scope of the present study.

Might the decreased phosphorylation and activity of othertargets for LKB1 be involved in neuronal degeneration?Importantly, deletion of both AMPK catalytic (a) subunits in thesame cells as those affected by LKB1 deletion failed to lead toneuronal degeneration (this study). Likewise, deletion of tuberoussclerosis complex 1 (TCS1), a downstream target of AMPK andinvolved in the control of the mammalian target of rapamycin(mTOR) and in regulating neuron polarity (Buckmaster et al., 2009;Swiech et al., 2008), using the RIP2-Cre transgene did not lead to

hind-limb paralysis (Mori et al., 2009b; Mori et al., 2009a). Thesefindings therefore argue against changes in mTOR signalling asbeing responsible for the axon degeneration observed here inLKB1KO mice. Indeed, we would note that, by activating themTOR pathway, LKB1 deletion might have been expected tomimic the effects of growth factors that act downstream of receptortyrosine kinases, such as nerve growth factor (Swiech et al., 2008),whereas we observed instead a degenerative phenotype, arguingstrongly against the involvement of such survival pathways in thiscase. Finally, Par1 (also known as MARK2) controls neurite growthand cell polarity (Biernat et al., 2002; Terabayashi et al., 2007), andhas been reported to lie downstream of LKB1 (Wang et al., 2007).However, although MARK2 knock-out mice displayed dwarfism,no obvious behavioural deficiencies were reported (Bessone et al.,1999). The latter observation again argues against the involvementof an LKB1-MARK2-tau signalling pathway in controlling axondevelopment in the spinal cord of LKB1KO mice.

We demonstrate here that LKB1 is essential to maintain normalpolarity and function of a crucial subset of neurons in the brain.Mice deleted for LKB1 in these cells might provide a useful animalmodel for studying axon degeneration and certainneurodegenerative disorders in humans.

METHODSGeneration of mutant mice lacking LKB1, or AMPKa1 andAMPKa2, in the spinal cordMice null for LKB1 (LKB1KO) (Sun et al., 2010b) and PRKAA1(AMPKa1 and a2; AMPKdKO) (Sun et al., 2010a) in the spinalcord, pancreatic -cell and a restricted group of hypothalamicneurons were generated as previously described. Neither LKB1heterozygous (LKB1fl/+), nor RIP2-Cre-recombinase-positive (wild-type) mice displayed symptoms of paralysis or abnormal spinal cordmorphology and were used as controls for LKB1KO micethroughout.

Mouse maintenance and dietMice were housed at two to five animals per cage in a pathogen-free facility with a 12-hour light-dark cycle. Animals were fed adlibitum with a standard mouse chow diet (Research Diet, NewBrunswick, NJ). LKB1KO mice were kept alive for 2 weeks afterthe initial onset of hind-limb dysfunction. Subsequently, mice wereculled by cervical dislocation. All in vivo procedures were approvedby the UK Home Office according to the Animals (ScientificProcedures) Act 1986 and were performed at the CentralBiomedical Service, Imperial College London, UK.

Body weight and foot print (paralysis) measurementsFed mice were weighed 1 and 2 weeks after the first onset ofparalysis. Weight increases after paralysis were calculated. Tomeasure locomotion by following foot prints, both fore-paws wereimmersed in red ink and hind-paws were immersed in blue inkbefore mice were allowed to walk freely on white filter paper(Whatman).

RNA extraction and qRT-PCRTotal cellular RNA from the cervical, thoracic and lumbar regionsof mouse spinal cord and hypothalamus was obtained using TRIzolreagent (Invitrogen, Paisley, UK) and RNA was further purified

Disease Models & Mechanisms 199

LKB1 and axon degeneration RESEARCH ARTICLE

Fig. 5. AMPKdKO mice do not develop hind-limb paralysis. (A)RT-PCRanalysis of AMPKa1 transcript levels and the effects of Cre expression onAMPKa2 transcript levels in different segments of the spinal cord of LKB1KOmice and their wild-type controls. For AMPKa1, the product sizes were 588and 408 bp for the wild-type and null alleles, respectively. NS, non-specificband; Cer, cervical; Thr, thoracic; Lum, lumbar segment of the spinal cord. Thecorresponding values for AMPKa2 were 588 and 375 bp. (B)RepresentativeH&E staining of the white matter of thoracic region of spinal cord fromAMPKdKO mice and their heterozygous controls at the age of 12-weeks old.Scale bar: 50m.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

against DNA contamination with a DNA-free kit (AppliedBiosystems, Warrington, UK). Total RNA (1.5-2 g) was thenreverse transcribed into cDNA with a high-capacity reverse tran-scription kit (Applied Biosystems, Warrington, UK) according tothe manufacturer’s instructions. To detect the deletion of LKB1exons 3-6, two pairs of primers within exon 1 (LKB1 fwd: 5�-AG-GTGAAGGAGGTGCTGG-3�) and 8 (LKB1 rev: 5�-TCT -GGGCTTGGTGGGATA-3�) were designed. To detect deletion ofthe allele encoding AMPKa1, the following primers were used:Ampka1-knockout_forward: 5�-CACCCTCAC ATCATCAAA-3�;Ampka1-knockout_reverse: 5�-AAACCACTCGTGTTCCCT-3�.The size of product generated from the wild-type allele was 588 bp,whereas that from the AMPKa1-knockout allele was 408 bp. Todetect the deletion of the allele encoding AMPKa2, the primersused were: Ampka2-knockout_forward: 5�-GTCTGCCGTGGAT-TACTG-3�; Ampka2-knockout_reverse: 5�-AGCTGGTCT -TGAGGGTCA-3�. The size of wild-type-allele-derived product was588 bp and that of the AMPKa2-knockout was 375 bp.

Western (immuno)blot analysisMouse spinal cord was snap frozen in liquid nitrogen before lysisin ice-cold buffer [in mM: 50 Tris-HCl (pH 7.4, 4°C), 150 NaCl, 1NaF, 1 Na3VO4, 1 ethylenediaminetetraacetic acid, 1 phenylmethyl-sulfonyl fluoride, 1% Nonidet P40, 0.25% sodium deoxycholate, andprotease inhibitor cocktail (Roche, Burgess Hill, UK)]. Protein (50g) extracted from cervical, thoracic and lumbar regions of thespinal cord were loaded onto SDS-PAGE gels (10% Tris-acrylamidegel) for analysis.

Immunohistochemistry, and H&E, LFB and PAS stainingDifferent segments of the spinal cord were fixed in 10% neutralformalin overnight and embedded in paraffin wax before sectioninginto slices of ~7 m [for immunofluorescent and diaminobenzidine(DAB) staining] or 12 m (for LFB and PAS staining). Slides weresubmerged sequentially in 100% [vol/vol] xylene followed bydecreasing concentrations of industrial methylated spirits to removeparaffin wax. Antigen epitopes were then retrieved (de-crosslinked)in Tris-EDTA-0.05% [vol/vol] Tween buffer (pH 9.0) or 10 mMsodium citrate buffer (pH 6.0). Slides were subsequently blockedin 5% [vol/vol] goat serum in Tris-buffered saline with 0.05%[vol/vol] Tween (TBS-T) for 20 minutes at room temperature andthen incubated overnight at 4°C in a mixture of primary antibodiesat the concentrations indicated. After being washed in TBS-T threetimes for 5 minutes each, slices blotted with primary antibodieswere either visualized with Alexa-Fluor-568- or -488-conjugatedIgG (1:500; Invitrogen, Paisley, UK) under fluorescence microscopyusing an Olympus IX-81 microscope (10� objective lens; datacollection with cell^R software) or further incubated withbiotinylated secondary antibodies (universal anti-mouse/anti-rabbitantibody) and subsequently revealed with peroxidase substrateusing DAB (Vector Laboratories, Peterborough, UK).

For H&E staining, rehydrated slices were submerged inhaematoxylin for 5 minutes. After being washed in water, sliceswere dipped in 1% [vol/vol] hydrogen chloride in 70% methanol[vol/vol] five times, followed by staining with 1% eosin [wt/vol] for30 seconds.

For LFB staining, de-waxed sections were immersed in 0.1% LFBsolution [wt/vol] (Sigma-Aldrich, Dorset, UK) at 60°C overnight.

After being washed subsequently in 95% methanol and distilledwater, slices were brought in 0.05% lithium carbonate [wt/vol] and70% methanol. After being washed in distilled water, slices werestained in 0.01% cresyl fast violet solution (Merck, Hoddesdon, UK)at 60°C for 10 minutes.

For PAS staining, de-waxed sections were oxidized in 1% periodicacid [vol/vol] for 5 minutes. After being washed in distilled water,slices were immersed in Schiff buffer (VWR, Lutterworth, UK) for10 minutes. Slices were subsequently counter-stained for nucleiwith haematoxylin for 1 minute.

AntibodiesAntibodies used in western blot analysis and immunohistochemistrywere: rabbit anti-LKB1 antibody, rabbit anti-tau antibody, mouseanti-GAPDH antibody (all from Millipore, Watford, UK), mouse anti--tubulin antibody (Sigma Aldrich, Dorset, UK), rabbit anti-Creantibody (Novagen, Nottingham, UK), rat anti-F4/80 antibody (AbDSerotec, Oxford, UK), and mouse anti-NF165kD (2H3)(Developmental Studies Hybridoma Bank, Iowa City, IA),

Statistical analysisData were expressed as means ± s.e.m. Significance was tested byappropriate unpaired or paired Student’s t-tests with Bonferronicorrections as required, or ANOVA using Graphpad 4.0. P<0.05was considered significant.ACKNOWLEDGEMENTSSupported by grants to G.A.R. from the Wellcome Trust (Programme Grant081958/2/07/Z), The European Union (FP6 ‘Save Beta’) and the Medical Research

dmm.biologists.org200

LKB1 and axon degenerationRESEARCH ARTICLE

Clinical issueThe molecular processes underlying axon degeneration in manyneurodegenerative diseases, including Alzheimer’s disease, Parkinson’s diseaseand multiple sclerosis, are only partially understood. Approaches that block orreverse these conditions are urgently needed. LKB1 (also known as STK11) is aprotein kinase and tumour suppressor with multiple functions; its downstreamtargets include AMP-activated protein kinase (AMPK) and sporadic Alzheimer’sdisease (SAD)-A/B kinase (also known as Brsk1/2). Studies in worms indicatethat LKB1 controls neuronal polarity by affecting axon differentiation, in partthrough interacting with SAD-A/B kinase. However, the role of LKB1 in themammalian CNS, and its potential involvement in the pathology ofneurodegenerative diseases, has not been well characterized.

ResultsIn this study, the authors investigate mice conditionally lacking LKB1 in thespinal cord, some parts of the brain and endocrine pancreas, and show thatLKB1 is essential in the CNS to prevent neurodegeneration and eventualparalysis and death in mice. At the cellular level, lack of LKB1 in neurons led toabnormal microtubule structures and a decrease in neurofilament and tubulinexpression. Surprisingly, one of the key downstream targets of this enzyme,AMPK, seems not to be the mediator of the required signals, whereas anotherLKB1 target, SAD-A/B kinase, and its subsequent phosphorylation of tauprotein are involved in preventing axonal degeneration.

Implications and future directionsThese results establish a role for LKB1 in promoting neuronal survival and thecontrol of motor function in an in vivo mammalian system. Approaches thatenhance the activity of LKB1 or its relevant downstream targets might providea novel therapeutic strategy for preventing or reversing the pathology ofneurodegenerative diseases.

doi:10.1242/dmm.007179

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Council (G0401641), and to I.L. and GAR from the Wellcome Trust (WT082366MA).We thank Lorraine Lawrence for the preparation of brain and spinal cord slices,Kate Hughes (Department of Veterinary Medicine, University of Cambridge, UK) forthe preparation and analysis of tissue slices, and Gabriela da Silva Xavier andMerewyn Loder for careful reading of the manuscript.

COMPETING INTERESTSThe authors declare no competing or financial interests.

AUTHOR CONTRIBUTIONSG.S. performed experiments and drafted the manuscript; R.R. and I.L. designedexperiments and edited the manuscript; G.A.R. designed the research and wrotethe manuscript.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.006833/-/DC1

REFERENCESArimura, N. and Kaibuchi, K. (2007). Neuronal polarity: from extracellular signals to

intracellular mechanisms. Nat. Rev. Neurosci. 8, 194-205.Barnes, A. P., Lilley, B. N., Pan, Y. A., Plummer, L. J., Powell, A. W., Raines, A. N.,

Sanes, J. R. and Polleux, F. (2007). LKB1 and SAD kinases define a pathway requiredfor the polarization of cortical neurons. Cell 129, 549-563.

Becerra, J. L., Puckett, W. R., Hiester, E. D., Quencer, R. M., Marcillo, A. E., Post, M. J.and Bunge, R. P. (1995). MR-pathologic comparisons of wallerian degeneration inspinal cord injury. Am. J. Neuroradiol. 16, 125-133.

Bessone, S., Vidal, F., Le, B. Y., Epelbaum, J., Bluet-Pajot, M. T. and Darmon, M.(1999). EMK protein kinase-null mice: dwarfism and hypofertility associated withalterations in the somatotrope and prolactin pathways. Dev. Biol. 214, 87-101.

Biernat, J. and Mandelkow, E. M. (1999). The development of cell processes inducedby tau protein requires phosphorylation of serine 262 and 356 in the repeat domainand is inhibited by phosphorylation in the proline-rich domains. Mol. Biol. Cell. 10,727-740.

Biernat, J., Gustke, N., Drewes, G., Mandelkow, E. M. and Mandelkow, E. (1993).Phosphorylation of Ser262 strongly reduces binding of tau to microtubules:distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11,153-163.

Biernat, J., Wu, Y. Z., Timm, T., Zheng-Fischhofer, Q., Mandelkow, E., Meijer, L. andMandelkow, E. M. (2002). Protein kinase MARK/PAR-1 is required for neuriteoutgrowth and establishment of neuronal polarity. Mol. Biol. Cell 13, 4013-4028.

Boudeau, J., Sapkota, G. and Alessi, D. R. (2003). LKB1, a protein kinase regulatingcell proliferation and polarity. FEBS Lett. 546, 159-165.

Buckmaster, P. S., Ingram, E. A. and Wen, X. (2009). Inhibition of the mammaliantarget of rapamycin signaling pathway suppresses dentate granule cell axonsprouting in a rodent model of temporal lobe epilepsy. J. Neurosci. 29, 8259-8269.

Buss, A. and Schwab, M. E. (2003). Sequential loss of myelin proteins during Walleriandegeneration in the rat spinal cord. Glia 42, 424-432.

Buss, A., Brook, G. A., Kakulas, B., Martin, D., Franzen, R., Schoenen, J., Noth, J.and Schmitt, A. B. (2004). Gradual loss of myelin and formation of an astrocytic scarduring Wallerian degeneration in the human spinal cord. Brain 127, 34-44.

Choudhury, A. I., Heffron, H., Smith, M. A., Al-Qassab, H., Xu, A. W., Selman, C.,Simmgen, M., Clements, M., Claret, M., Maccoll, G. et al. (2005). The role of insulinreceptor substrate 2 in hypothalamic and beta cell function. J. Clin. Invest. 115, 940-950.

Coleman, M. P. and Perry, V. H. (2002). Axon pathology in neurological disease: aneglected therapeutic target. Trends Neurosci. 25, 532-537.

de Anda, F. C., Pollarolo, G., Da Silva, J. S., Camoletto, P. G., Feiguin, F. and Dotti, C.G. (2005). Centrosome localization determines neuronal polarity. Nature 436, 704-708.

Dotti, C. G., Sullivan, C. A. and Banker, G. A. (1988). The establishment of polarity byhippocampal neurons in culture. J. Neurosci. 8, 1454-1468.

Fujimura, M., Usuki, F., Sawada, M. and Takashima, A. (2009). Methylmercuryinduces neuropathological changes with tau hyperphosphorylation mainly throughthe activation of the c-jun-N-terminal kinase pathway in the cerebral cortex, but notin the hippocampus of the mouse brain. Neurotoxicology 30, 1000-1007.

Gannon, M., Shiota, C., Postic, C., Wright, C. V. and Magnuson, M. (2000). Analysisof the Cre-mediated recombination driven by rat insulin promoter in embryonic andadult mouse pancreas. Genesis 26, 139-142.

Garbern, J. Y. (2005). Pelizaeus-Merzbacher disease: pathogenic mechanisms andinsights into the roles of proteolipid protein 1 in the nervous system. J. Neurol. Sci.228, 201-203.

Hawley, S. A., Boudeau, J., Reid, J. L., Mustard, K. J., Udd, L., Makela, T. P., Alessi, D.R. and Hardie, D. G. (2003). Complexes between the LKB1 tumor suppressor,STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activatedprotein kinase cascade. J. Biol. 2, 28.

Hennige, A. M., Burks, D. J., Ozcan, U., Kulkarni, R. N., Ye, J., Park, S., Schubert,M., Fisher, T. L., Dow, M. A., Leshan, R. et al. (2003). Upregulation of insulinreceptor substrate-2 in pancreatic beta cells prevents diabetes. J. Clin. Invest. 112,1521-1532.

Hirokawa, N. and Takemura, R. (2005). Molecular motors and mechanisms ofdirectional transport in neurons. Nat. Rev. Neurosci. 6, 201-214.

Jansen, M., Ten Klooster, J. P., Offerhaus, G. J. and Clevers, H. (2009). LKB1 andAMPK family signaling: the intimate link between cell polarity and energymetabolism. Physiol. Rev. 89, 777-798.

Kim, J. S., Hung, W., Narbonne, P., Roy, R. and Zhen, M. (2010). C. elegans

STRADalpha and SAD cooperatively regulate neuronal polarity and synapticorganization. Development 137, 93-102.

Kimura, T., Watanabe, H., Iwamatsu, A. and Kaibuchi, K. (2005). Tubulin and CRMP-2complex is transported via Kinesin-1. J. Neurochem. 93, 1371-1382.

Kishi, M., Pan, Y. A., Crump, J. G. and Sanes, J. R. (2005). Mammalian SAD kinases arerequired for neuronal polarization. Science 307, 929-932.

Leenen, P. J., de Bruijn, M. F., Voerman, J. S., Campbell, P. A. and van Ewijk, W.(1994). Markers of mouse macrophage development detected by monoclonalantibodies. J. Immunol. Methods 174, 5-19.

Lefcort, F. and Bentley, D. (1989). Organization of cytoskeletal elements andorganelles preceding growth cone emergence from an identified neuron in situ. J.

Cell Biol. 108, 1737-1749.Lewis, J., McGowan, E., Rockwood, J., Melrose, H., Nacharaju, P., Van Slegtenhorst,

M., Gwinn-Hardy, K., Paul, M. M., Baker, M., Yu, X. et al. (2000). Neurofibrillarytangles, amyotrophy and progressive motor disturbance in mice expressing mutant(P301L) tau protein. Nat. Genet. 25, 402-405.

Lin, W. L., Zehr, C., Lewis, J., Hutton, M., Yen, S. H. and Dickson, D. W. (2005).Progressive white matter pathology in the spinal cord of transgenic mice expressingmutant (P301L) human tau. J. Neurocytol. 34, 397-410.

Lin, X., Taguchi, A., Park, S., Kushner, J. A., Li, F., Li, Y. and White, M. F. (2004).Dysregulation of insulin receptor substrate 2 in beta cells and brain causes obesityand diabetes. J. Clin. Invest. 114, 908-916.

Lizcano, J. M., Goransson, O., Toth, R., Deak, M., Morrice, N. A., Boudeau, J.,Hawley, S. A., Udd, L., Makela, T. P., Hardie, D. G et al. (2004). LKB1 is a masterkinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1.EMBO J. 23, 833-843.

Mori, H., Inoki, K., Munzberg, H., Opland, D., Faouzi, M., Villanueva, E. C., Ikenoue,T., Kwiatkowski, D., Macdougald, O. A., Myers, M. G., Jr et al. (2009a). Critical rolefor hypothalamic mTOR activity in energy balance. Cell Metab. 9, 362-374.

Mori, H., Inoki, K., Opland, D., Muenzberg, H., Villanueva, E. C., Faouzi, M.,Ikenoue, T., Kwiatkowski, D., Macdougald, O. A., Myers, M. G., Jr et al. (2009b).Critical roles for the TSC-mTOR pathway in -cell function. Am. J. Physiol. Endocrinol.

Metab. [Epub ahead of print] doi:10.1152/ajpendo.00262.2009.Perrin, F. E., Lacroix, S., Aviles-Trigueros, M. and David, S. (2005). Involvement of

monocyte chemoattractant protein-1, macrophage inflammatory protein-1alphaand interleukin-1beta in Wallerian degeneration. Brain 128, 854-866.

Remedi, M. S., Rocheleau, J. V., Tong, A., Patton, B. L., McDaniel, M. L., Piston, D.W., Koster, J. C. and Nichols, C. G. (2006). Hyperinsulinism in mice withheterozygous loss of K(ATP) channels. Diabetologia 49, 2368-2378.

Shelly, M., Cancedda, L., Heilshorn, S., Sumbre, G. and Poo, M. M. (2007). LKB1/STRADpromotes axon initiation during neuronal polarization. Cell 129, 565-577.

Sobolev, S. M. (1959). A histochemical study of certain periodic acid-schiff-positivesubstances in the macrophages. Bull. Exp. Biol. Med. 47, 633-637.

Sun, G., Tarasov, A. I., McGinty, J., McDonald, A., da Silva Xavier, G., Gorman, T.,Marley, A., French, P. M., Parker, H., Gribble, F. et al. (2010a). Ablation of AMP-activated protein kinase alpha1 and alpha2 from mouse pancreatic beta cells andRIP2.Cre neurons suppresses insulin release in vivo. Diabetologia 53, 924-936.

Sun, G., Tarasov, A. I., McGinty, J. A., French, P. M., McDonald, A., Leclerc, I. andRutter, G. A. (2010b). LKB1 deletion with the RIP.Cre-transgene modifies pancreatic-cell morphology and enhances insulin secretion in vivo. Am. J. Physiol. Endocrinol.

Metab. 298, E1261-E1273.Sutherland, C. M., Hawley, S. A., McCartney, R. R., Leech, A., Stark, M. J., Schmidt,

M. C. and Hardie, D. G. (2003). Elm1p is one of three upstream kinases for theSaccharomyces cerevisiae SNF1 complex. Curr. Biol. 13, 1299-1305.

Swiech, L., Perycz, M., Malik, A. and Jaworski, J. (2008). Role of mTOR in physiologyand pathology of the nervous system. Biochim. Biophys. Acta. 1784, 116-132.

Terabayashi, T., Itoh, T. J., Yamaguchi, H., Yoshimura, Y., Funato, Y., Ohno, S. andMiki, H. (2007). Polarity-regulating kinase partitioning-defective 1/microtubuleaffinity-regulating kinase 2 negatively regulates development of dendrites onhippocampal neurons. J. Neurosci. 27, 13098-13107.

Wang, J. W., Imai, Y. and Lu, B. (2007). Activation of PAR-1 kinase and stimulation oftau phosphorylation by diverse signals require the tumor suppressor protein LKB1. J.

Neurosci. 27, 574-581.

Disease Models & Mechanisms 201

LKB1 and axon degeneration RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

Wicksteed, B., Brissova, M., Yan, W., Opland, D. M., Plank, J. L., Reinert, R. B.,Dickson, L. M., Tamarina, N. A., Philipson, L. H., Shostak, A. et al. (2010).Conditional gene targeting in mouse pancreatic beta cells: analysis of ectopic Cretransgene expression in the brain. Diabetes [Epub ahead of print] doi: 10.2337/db10-0624.

Woods, A., Johnstone, S. R., Dickerson, K., Leiper, F. C., Fryer, L. G., Neumann, D.,Schlattner, U., Wallimann, T., Carlson, M. and Carling, D. (2003). LKB1 is theupstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13, 2004-2008.

Zhang, L., Li, J., Young, L. H. and Caplan, M. J. (2006). AMP-activated protein kinaseregulates the assembly of epithelial tight junctions. Proc. Natl. Acad. Sci. USA 103,17272-17277.

Zhang, Z. and Guth, L. (1997). Experimental spinal cord injury: Wallerian degenerationin the dorsal column is followed by revascularization, glial proliferation, and nerveregeneration. Exp. Neurol. 147, 159-171.

Zhao, C., Takita, J., Tanaka, Y., Setou, M., Nakagawa, T., Takeda, S., Yang, H. W.,Terada, S., Nakata, T., Takei, Y. et al. (2001). Charcot-Marie-Tooth disease type 2Acaused by mutation in a microtubule motor KIF1Bbeta. Cell 105, 587-597.

dmm.biologists.org202

LKB1 and axon degenerationRESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M