REVIEWS Nuclear and cytosolic JNK signalling in neurons Eleanor T. Coffey

Abstract | It has been over 20 years since JUN amino‑terminal kinases (JNKs) were identified as protein kinases that are strongly activated by cellular stress and that have a key role in apoptosis. Examination of Jnk-knockout mice and characterization of JNK behaviour in neuronal cells has further revealed the importance of the JNK family in the nervous system. As well as regulating neuronal death, JNKs govern brain morphogenesis and axodendritic architecture during development, and regulate important neuron-specific functions such as synaptic plasticity and memory formation. This Review examines the evidence that the spatial segregation of JNKs in neurons underlies their distinct functions and that compartment-specific targeting of JNKs may offer promising new therapeutic avenues for the treatment of diseases of the nervous system, such as stroke and neurodegenerative disorders.

Turku Centre for Biotechnology, Åbo Akademi University and the University of Turku,Tykistokatu 6, Turku 20520, Finland. Correspondence to E.T.C. e‑mail: [email protected] doi:10.1038/nrn3729

JUN amino‑terminal kinases (JNKs; also known as stress-activated protein kinases (SAPKs1)) transfer phosphate groups to serine or threonine residues that are flanked by a carboxy‑terminal proline. JNKs contrib‑ ute to the high levels of proline-directed phosphoryla‑ tion that is peculiar to the mammalian brain2,3. Indeed, JNK activity is higher in the brain than in any other mammalian tissue4–9, suggesting that members of this kinase family are key regulators of protein function in the nervous system. JNKs are components of a classical mitogenactivated protein kinase (MAPK) signalling cascade (FIG.  1a) that serves to filter noise and allow signal amplification while maintaining upstream kinase com‑ plexity, enabling signalling diversity. JNKs and the protein scaffolds to which they bind display distinct patterns of subcellular compartmentalization in neu‑ rons that probably underlie their pleiotropic actions, which fall into two main categories: physiological and stress-inducible. Studies of Jnk-knockout (Jnk–/–) mice have revealed roles for JNKs in brain morpho‑ genesis, neuronal pathfinding, axodendritic architec‑ ture maintenance and neuronal death after excitotoxic insults (TABLE 1). JNKs have also received considerable attention in the context of neurodegenerative diseases and, more recently, human genetics data have impli‑ cated the JNK pathway in the pathophysiology of neu‑ ropsychiatric disorders10–15. Here, I review the effects of JNKs on brain development, the adult brain and

patho­logical states, taking into account how compart‑ mental segregation of JNKs in neurons enables them to exert diverse functions. For the purposes of this article, JNK1, JNK2 and JNK3 are referred to as JNK isoforms (that is, being derived from closely related but distinct genes), whereas the splice variants of each JNK will be referred to as splice variants (although strictly they are also isoforms). Throughout, ‘JNK’ and ‘JNKs’ refer to the family of JNK kinases.

JNK mRNA and protein distribution in the brain Three JNK genes are expressed in the human brain, and together they generate ten splice variants that are nearly all either 46 kDa or 54 kDa in size1 (FIG. 1b). Studies of mRNA in mice have shown that Jnk1 (also known as Mapk8) and Jnk2 (also known as Mapk9) are ubiquitously expressed throughout the body, whereas expression of Jnk3 (also known as Mapk10) is largely restricted to the brain, with low levels of Jnk3 mRNA also found in the testes16. The highest levels of Jnk1, Jnk2 and Jnk3 mRNA are found in the neocortex, closely followed by the hip‑ pocampus, thalamus and midbrain. Jnk1 is developmen‑ tally regulated: high levels of Jnk1 mRNA are expressed in the developing rat brain, and these decline postnatally, although Jnk1 mRNA levels remain high in the olfactory area throughout adulthood17. In situ hybridization studies in the mouse and rat suggest that Jnk3 mRNA is the most highly expressed Jnk transcript in the adult brain followed by Jnk2 mRNA and then Jnk1 mRNA17,18.

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REVIEWS a

MEKKs

DLK

MKKs

MAPKs

TNIK

JNK1

TAOK2

MEKK1

MKK4

MKK7

JNK2

MLKs

ASK1

MKK3 or MKK6

JNK3

p38

Nuclear and/or cytoplasmic substrates

b JNK1

JNK2

JNK3 3α1 3α2

c

MEKK4

1α1 1α2 1β1 1β2

C

2α1 2α2 2β1 2β2

C

Cerebral cortex I/II III Cortical IV V layers VI

C

C

C C

C

C

C Hippocampus

Cerebellum

CA1

CA3 CA4

C

Purkinje cell layer Granule cell layer

JNK1

JNK2

JNK3

Nature | Neuroscience Figure 1 | The JNK cascade and JNK isoform expression in Reviews the mammalian brain.  a | JUN amino‑terminal kinases (JNKs) belong to a classical mitogen-activated protein kinase (MAPK) signalling cascade. JNKs are activated by dual phosphorylation of the TPY motif within their activation loop by two MAPK kinases (MKKs): namely, MKK4 and MKK7. Various MKK kinases (MEKKs) phosphorylate and thereby activate MKK7 and/or MKK4 (and other MKKs). Indeed, MEKKs comprise an overwhelmingly complex group of signalling molecules1. MEKK1, MEKK4, MLK1–MLK3, apoptosis signal-regulating kinase 1 (ASK1), thousand-and-one amino acid kinase 2 (TAOK2), TRAF2 and NCK-interacting protein kinase (TNIK) and dual leucine zipper bearing kinase (DLK) are all MEKKs that are known to have important functions in the nervous system15,103,178–182. b | In the brain, JNK1, JNK2 and JNK3 are alternatively spliced to yield ten proteins, which nearly all fall into two molecular weight categories of 54k Da and 46 kDa. The schematic depicts the ten JNK protein products, with homologous protein regions indicated by the coloured blocks. The kinase domain (blue) includes 11 subdomains. Alternative splicing in subdomains IX and X (indicated in light blue) gives rise to α- and β-JNK variants, whereas alternative splicing at the carboxyl terminus yields long (54 kDa) and short (46 kDa) JNK variants. c | An overview of JNK isoform protein expression in the mouse brain19,20. The subcellular distribution of JNK1 and JNK3 differs in the cortex, hippocampus and cerebellum. In the cortex and hippocampus, JNK1 expression is largely cytosolic and found in the neuropile, whereas JNK3 expression is predominantly nuclear and found in approximately 30% of cells in layers III and V. Of note, JNK3 is the dominant JNK isoform expressed throughout the hippocampus, where it is found in nuclei of approximately 90% of neurons. In the cerebellum, JNK3 is found in nuclei of Purkinje cells and cerebellar granule neurons, whereas JNK1 expression is prominent in the Purkinje cell dendrites. JNK2 expression is similar to JNK1 and JNK3 expression in the cortex, and this isoform is found in both the nuclei and somata of neurons in the hippocampus and cerebellum.

The mRNA expression patterns described above correlate with JNK protein levels in rodents19. A sum‑ mary of the regional distribution of JNK isoform expression in the adult mouse19,20 is shown in FIG. 1c. JNK1 and JNK3 show distinct subcellular distributions. JNK3 is prominent in the nuclei of Purkinje and gran‑ ule cells in the cerebellum and in the nuclei of a subset of neurons (~30%) in layers III and V of the cortex, which harbour projecting pyramidal neurons. By con‑ trast, JNK1 expression dominates in the somal cyto‑ plasmic space and neurofibres (axons and dendrites) in the cortex; it is conspicuously high in the cytosol, particularly in the dendrites, of Purkinje and thalamic neurons20. In the hippocampus, JNK3 expression is found in ~90% of pyramidal layer neurons, whereas JNK1 expression is restricted to the CA3, CA4 and the hilus of the dentate gyrus20. JNK1 expression is largely extranuclear in isolated cerebellar granule neurons, with the 46 kDa isoforms of JNK1 showing the highest levels of expression. The low levels of JNK1 expression that are found in the nuclei of these cells mostly com‑ prise the 54 kDa JNK1 variants21. In contrast to JNK1 and JNK3 expression, JNK2 expression is comparatively low in most brain regions and is distributed in both the cytosol and nuclei19–21. Compensatory increases in JNK isoform expression have been reported in Jnk−/− mice: JNK1 levels are increased in brain tissue from Jnk2−/− mice, whereas JNK2 levels are increased in the brains of Jnk3−/− mice19,22. Data from mice in which individual JNK genes were knocked out suggest that JNK1 activity accounts for most physiological JNK activity in the cor‑ tex and cerebellum, whereas JNK3 activity accounts for most JNK activity in the hippocampus and striatum9,19.

Subcellular division of JNK signalling Neurons are exquisitely complex in their subcellular com‑ partmentalization, hosting not only the generic nuclear and cytoplasmic compartments but also functionally specialized compartments with distinct morphologies and protein compositions (for example, axons, dendrites and dendritic spines). In the cytoplasm. The cytosolic nature of active JNK in neurons has been emphasized using various methodolog‑ ical approaches. Upon fractionation of forebrain extracts, active JNK separates to the postnuclear membrane frac‑ tion, with no trace of active kinase in the soluble frac‑ tion9. Consistent with this finding, antibodies raised against JNKs in their active state reveal a characteristi‑ cally punctate staining pattern in peripheral nervous system and CNS neurons, indicating that JNK associ‑ ates with vesicular structures9,23. Similarly, microporous filter-based separation of neurites and somata showed that active JNK predominates in neurites24. Accordingly, several physiological JNK substrates — namely, the microtubule-associated proteins (MAPs) MAP1B, MAP2 and superior cervical ganglion 10 protein (SCG10; also known as STMN2) — reside in the cytoplasm 6,7,25–30 (FIG. 2; see Supplementary information S1 (table)). The recent development of fluorescence resonance energy transfer sensors (FRET sensors) has provided improved

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REVIEWS Table 1 | Cellular and behavioural phenotypes of JNK-knockout animal models Model

Viability

Cellular or behavioural phenotype

Jnk1 mouse

Viable

• Disorganized cell positioning in cortical layers30 • Increased multipolar and bipolar cell motility rate30 • Decreased duration of multipolar stage30 • Decreased cell cycle exit in the embryonic brain30 • Anterior commissure (anterior and posterior) defect. Tracts degenerate from P12 onwards in mice26 • Loss of microtubule integrity in axons of P7 mice26 • Reduced microtubule stability in the embryonic brain30 • Disrupted microtubule integrity in the dendrites of the hippocampus from 3 months onwards26 • Disrupted dendrite architecture of Purkinje cells in adult mice7 • Absence of autotomy (self-mutilation) of degenerating limb after axotomy187 • Increased explorative behaviour188

Jnk2–/– mouse

Viable189

• Decreased dopaminergic cell loss in mouse MPTP Parkinson’s disease model190 • Loss of stress-induced learning impairment191 • Impaired long-term potentiation22

Jnk1–/–Jnk2–/– mouse

Embryonic lethal E11.5

• Impaired neural tube closure16,72 • Decreased cell death in the hindbrain at E9.0 (REFS 16,72)

–/–

186

(REFS 16,72)

Jnk3–/– mouse

Viable52

• Decreased cell damage in the hippocampus (CA1 and CA3) after excitotoxic insult (by kainate)52 • Decreased dopaminergic cell loss in mouse MPTP Parkinson’s disease model190 • Loss of stress-induced learning impairment191 • Reduced tonic-clonic seizures after weak (30 mg per kg) kainate exposure19 • Absence of autotomy (self-mutilation) of degenerating limb after axotomy187

Jnk2–/–Jnk3–/– mouse

Viable16,72

• Decreased dopaminergic cell loss in mouse MPTP Parkinson’s disease model190

Jnk1–/–Jnk2–/–Jnk3–/– mouse neurons

Viable in culture97

• Increased lifespan in vitro97 • Increased autophagy in neurons97 • Purkinje cell axons show increased cross-sectional area97 • Reduced number of dendrites in Purkinje cells97

basket-knockout (JNK homologue) Drosophila melanogaster

Embryonic lethal78

• Dorsal closure defect due to defective movement of epithelial layers, resulting in dorsal cuticle phenotype78

jnk‑1–/– Caenorhabditis Viable193 elegans

• Axonal transport defect leading to mislocalization of synaptic vesicle proteins118 • Prolonged retention of olfactory adaptation and salt chemotaxis learning (forgetting is impaired)192

E, embryonic day; JNK, JUN amino-terminal kinase; MPTP, 1‑methyl‑4‑phenyl-1,2,3,6-tetrahydropyridin; P, postnatal day.

Fluorescence resonance energy transfer sensors (FRET sensors). Fluorescence resonance energy transfer reporters that detect protein– protein interactions. Here, I refer to a tandem FRET sensor, which transfers light energy of a particular wavelength emitted by a donor fluorophore to an acceptor fluorophore to yield a FRET response that can be harnessed to provide spatiotemporal information on various functional readouts in living cells (for example, kinase activity).

spatial information on catalytic JNK activity 31,32. These cytosolic reporters confirm that JNK is active in neurites (albeit this finding was in differentiated NIE‑115 cells24) and they may provide a valuable means to monitor JNK activity with spatial and temporal resolution. In the cytoplasm, JNKs are partly located with mito‑ chondria33. This relationship has been examined in the context of neuronal death, as there is a net increase in mitochondria-localized JNK after exposure to excito‑ toxic stress34,35. This translocation of JNKs to mitochon‑ dria facilitates phosphorylation of BH3‑only members of the BCL‑2 protein family, BIMEL (BCL‑2‑interacting mediator of cell death extra long) and DP5 (also known as HRK)36–41 (Supplementary information S1 (table)), which are essential initiators of programmed cell death following exposure to noxious stimuli. JNK translocation occurs in response to trophic depriva‑ tion stress, activation of the p75 neurotrophin recep‑ tor and transient focal cerebral ischaemia, and leads to BAX-dependent release of cytochrome c and apoptotic death38,40.

The case for JNK being localized in dendritic spines is less clear. JNKs phosphorylate postsynaptic density pro‑ tein 95 (PSD95) and AMPA glutamate receptor (AMPAR) subunits42–44 (FIG. 2d; see Supplementary information S1 (table)), which are enriched in spines. JNK-interacting protein  3 (JIP3), a scaffolding protein that coordi‑ nates JNK activation45, is also found in spine heads46,47. Nonetheless, a proteomics analysis of PSDs failed to detect JNKs in this compartment, although upstream regulators of these kinases — MINK1 (misshapen-like kinase 1), TNIK (TRAF2 and NCK-interacting protein kinase) and MKK4 (also known as MAP2K4) — were identified48–50. Despite this lack of direct evidence that JNKs are active in spines, the functional changes exerted by JNKs at the PSD suggest that a closer look at whether JNKs are present in spines, and their functional impact in this location, may be warranted. In the nucleus. JNKs are not static in a given cellular compartment; for example, JNK3 translocates from the cytoplasm to the nucleus in pyramidal neurons of the

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REVIEWS Dendrite arborization MT stability

Dendrite P P P P

a

MT stabilization

MT

MT plasticity

P P SCG10 P MKK7

Mkk7 mRNA

Axon

b P P P P

P P P P

MAP2

P

PSD95

JNK1 MAP1B P

JNK1

c

AMPAR

P

d

JNK1

JNK1

JNK scaffold

P ?

P

Nucleus Poly(A)

P

P

P JNK1 Synaptic plasticity

Growth cone

Figure 2 | JNK function in the cytoplasm.  JUN amino-terminal kinase (JNK) phosphorylates various proteins that are Nature Reviews | Neuroscience involved in axonal growth and pathfinding (microtubule (MT)-associated protein 1B (MAP1B) and superior cervical 9,26 7,15 ganglion 10 protein (SCG10)) , dendrite arborization (MAP2) and synaptic plasticity (AMPA glutamate receptor (AMPAR) and postsynaptic density protein 95 (PSD95))42,43. a | JNK1 regulates MT stability via phosphorylation of substrates such as SCG10 and MAP1B26,30. SCG10 is a tubulin-sequestering protein that controls MT catastrophe events. SCG10 function is required for growth cone extension183, and phosphorylation of SCG10 on serine 62 and serine 73 by JNK1 stabilizes MTs and promotes migration30. Mkk7 mRNA is found in neuronal growth cones, where it may be locally translated to regulate axonal growth24. JNK scaffold proteins are found in the cytoplasm, where they confer a higher level of diversity to JNK signalling by tethering JNKs to different subcellular compartments. b | JNK1 phosphorylates MAP1B26, a protein that is important for stabilization of axonal MTs. The axons in Jnk1‑knockout mice display signs of degeneration26, and MAP1B is probably a mediator of this degeneration. c | The dendritic MAP high-molecular-weight MAP2 is also phosphorylated by JNK1. This modification is proposed to stabilize MTs and regulate dendritic arborization7,26,27. d | Dendritic spine proteins PSD95, and the AMPAR subunits GluR2 (long splice form) and GluR4 are phosphorylated by JNK1, possibly altering synaptic strength42,43. It remains unclear whether JNKs phosphorylate dendritic spine proteins inside or outside spines (denoted by the question mark).

Arborization A term used to describe the branching or ramification of dendrites.

Excitotoxicity A type of pathological neuronal death that results from excessive stimulation of glutamate receptors.

hippocampus in response to hypoxia and/or ischaemic injury 51. Stressors — such as excitotoxicity, withdrawal of trophic support or axotomy — trigger JNK-dependent phosphorylation of JUN and activating transcription factor 2 (ATF2) in the nucleus19,21,23,38,52–54. In turn, JNKphosphorylated JUN–ATF2 dimers positively regulate the Jun promoter, increasing JUN mRNA and protein levels and signalling cell death55–57 (FIG. 3a). This tran‑ scriptional trigger induces expression of the pro-apo‑ ptotic genes MAPK phosphatase 1 (Mkp1; also known as dual specificity phosphatase 1), Dp5, Bim and Puma (also known as Bbc3), which are apoptotic facilitators of the BCL‑2 protein family 36,38,58–62 and have been shown to contribute to the death of sympathetic neurons (in the absence of trophic support)63, cortical neurons (follow‑ ing excitotoxicity)64 and dopaminergic neurons (after axotomy)53 (FIG. 3b). The evidence that JUN transactivation triggers cell death comes from experiments in JUN‑AA mice — in which JUN serine 63 and serine 73 (which activate tran‑ scription when they are phosphorylated) are mutated to alanines, resulting in resistance to kainate-induced apoptosis — and in neurons — in which expression of a dominant-negative JUN was neuroprotective53,63,64. Whereas pharmacological inhibition of JNK is associ‑ ated with a broadly neuroprotective profile, a crucial role for JUN in mediating JNK-dependent neuronal

death has not been unequivocally demonstrated across all models28,41, and JNK is not always involved in JUNmediated death65. It may be that entirely independent gene regulatory events contribute to JNK-mediated apoptosis. Indeed, additional gene targets of MLK and JNK signalling were recently identified in sympathetic neurons deprived of trophic support. These include the endoplasmic reticulum stress pathway genes Trib3 (trib‑ bles homologue 3), Ddit3 (DNA damage-inducible tran‑ script 3) and Txnip (thioredoxin interacting protein), a regulator of oxidative stress, among others62,66 (FIG. 3a). Although the precise mechanisms of JNK-mediated apoptosis remain to be elucidated, it is notable that these genes encode proteins that are enriched in the nucleus. Each is a regulator of gene expression, thereby empha‑ sizing the importance of the nucleus as a location for stress-triggered events62. A major breakthrough in understanding nuclear JNK function emerged from recent studies showing that JNK directly targets chromatin modifiers, driving histone phosphorylation and acetylation67–70. In this way, JNKs can elicit a more profound influence on gene expres‑ sion than was previously expected, not only in stress responses but also during neuronal differentiation. JNK2 and JNK3 phosphorylate serine 10 of histone H3 in vitro, an event that is associated with the relaxation of chromatin and active gene transcription67,70 (FIG. 3c). JNK

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REVIEWS a Stress P

Stress P

P JNK3

P

• Bim • Jund • Mkp1 • Puma • Trib3 • Txnip

AP1

JNK2

P

P JNK2 or JNK3

P P P P

Apoptosis

P P P P Jun JUN ATF2 Nucleus

b

c Nucleus P

P

P

P JNK1

JNK MKP1 P P P P Mkp1 JUN ATF2

Axon growth and branching

Nucleus P

P JNK2 or JNK3

RNA Pol II

• G3bp1 • Ppp2r • Pabpn1 • Traf2

P Histone H3

Differentiation

Figure 3 | The nuclear function of JNKs.  JUN amino-terminal kinase (JNK) signalling in Nature Reviews | Neuroscience the nucleus is involved in neuronal stress responses as well as developmental processes. a | Under various stress conditions, active isoforms of JNK in the nucleus induce the AP1 stress response directly or through an increase in the levels of AP1 family of transcription factors from the JUN and ATF family. The AP1‑responsive apoptotic death genes mitogen-activated protein kinase phosphatase 1 (Mkp1), jun D proto-oncogene (Jund), thioredoxin interacting protein (Txnip), Puma (an apoptosis facilitator) and BCL‑2‑interacting mediator of cell death (Bim) are examples of genes that are upregulated in response to trophic withdrawal stress62. JNKs can translocate to the nucleus following certain stresses; for example, JNK3 translocates to the nucleus in response to cerebral hypoxic stress135. b | Mkp1 expression is induced by JNKs in response to trophic deprivation stress62. MKP1 in the cytoplasm acts to negatively regulate JNK1 catalytic activity and control axon branching during cortical development184. c | JNKs regulate chromatin modifiers; for example, JNK2 and JNK3 phosphorylate histone H3 on serine 10, which relaxes chromatin structure67. This occurs in differentiating neurons, in which catalytically active JNK1 and JNK3 are found bound to the active promoters for GTPase activating protein binding protein 1 (G3bp1), Ppp2r (protein phosphatase 2 regulatory subunit genes), poly(A) binding protein nuclear 1 (Pabpn1) and TNF receptorassociated factor 2 (Traf2)67. RNA Pol II, RNA polymerase II; Trib3, tribbles homologue 3.

AP1 (Activating protein1). AP1 is a transcription factor dimer comprising proteins belonging to JUN, ATF or FOS families.

Exencephaly A developmental defect in which the brain extrudes outside the skull.

Neurulation A process during early development of the CNS in which the neural plate is formed. It is followed by neural plate closure and formation of the neural tube.

also interacts with the ATAC histone acetyltransferase complex in Drosophila melanogaster and facilitates the expression of JNK target genes under resting condi‑ tions but represses their expression following osmotic stress by inhibiting upstream activators71. During the differentiation of mouse stem cells to neurons, JNK associates with active promoters enriched with bind‑ ing motifs for the nuclear transcription factor Y (NFY); surprisingly, this study found little enrichment of JNK at AP1‑binding motifs, although JUN and ATF are the best-studied nuclear targets of JNK67. The genes bound by JNK in differentiating neurons include those involved in functions such as nervous system develop‑ ment, the cell cycle, gene expression and cell death67. It remains to be seen whether chromatin immunopre‑ cipitation (ChIP) analysis using stressed neurons reveals JNK binding at AP1‑binding motifs, which would be in support of earlier work indicating that AP1 is a hallmark of neuronal stress53,63,64.

JNK function in brain development Brain morphogenesis and developmental death. The phenotypes observed in JNK-knockout mice high‑ lighted the importance of JNK function in the nervous system (TABLE 1). Two research groups simultaneously generated JNK-knockout mice using independent transgenic strategies16,72. Jnk3−/− mice show no obvious developmental brain defects and survive normally, but mice lacking both Jnk1 and Jnk2 (Jnk1−/−Jnk2−/− mice) die between embryonic day 11 (E11) and E12 (REF. 16). The Jnk1−/−Jnk2−/− mice fail to complete neural tube clo‑ sure, displaying an overt exencephaly phenotype. They exhibit region-specific alterations in cell survival, with decreased apoptosis in the hindbrain neuroepithelium just before neural tube closure and increased apoptosis and caspase 3 activation in the forebrain16,72 (FIG. 4A). Neurulation involves regulated cell death and prolifera‑ tion as well as coordinated changes in cell shape and cell movement. No defects in neural crest cell migra‑ tion were detected in the Jnk1−/−Jnk2−/− mice, but later studies implicated JNKs in the migration of various cell types73. Similarly, cell proliferation was unchanged in these mice16,72, and the exencephaly phenotype is considered to be a direct consequence of deregulated apoptosis. The neurulation defect was gene-dosedependent and the presence of only one Jnk1 allele was sufficient to maintain viability and an intact neu‑ ral tube72. Of note, compound knockout of Jnk1 and Jnk3, or Jnk2 and Jnk3, does not result in neural tube defects16. Interestingly, the exencephaly phenotype was not reported upon genetic ablation of Mkk4 or Mkk7 (also known as Map2k7), which encodes an upstream regulator of JNKs, and compound disruption of both of the genes results in severe growth retardation and embryonic lethality at E9.5, before the completion of neurulation74–76. However, in flies, disruption of hemipterous (the fly homologue of MKK7) results in failed dorsal closure, as does disruption of basket, the JNK homologue77,78. Moreover, MKKs of the JNK pathway are known to regulate morphogenic movements dur‑ ing development in a range of organisms. For example, members of this family regulate convergent extension in zebrafish (Mkk4b) and Xenopus laevis (mkk7) and dorsal ventral patterning in zebrafish (Mkk4a)76,79–81. It is striking that among the substrates of JNK (Supplementary information S1 (table)), only one phen‑ ocopies Jnk1−/−Jnk2−/− mice when it is genetically deleted. Indeed, mice lacking Jun, the transcription factor target of JNK, die from heart malformations at E12.5, with increased apoptosis in the liver; however, they show no cranial defects82. Similarly, mice lacking the JNK target Atf2 undergo normal cranial morphogenesis and survive until birth, after which they die from respiratory failure83. Only MARCKS-like protein 1 (MARCKSL1; also known as MacMARCKS), an actin regulatory protein belong‑ ing to the GMC family 84, identified in a screen for novel JNK targets85, causes neural tube defects upon knockout. Indeed, genetic deletion of MARCKSL1 results in exen‑ cephaly 86,87. JNK phosphorylates MARCKSL1 at three residues, and this leads to increased actin bundling and retarded migration85.

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REVIEWS A Morphogenesis

Ba Pathfinding axon stability Control

Wild-type Jnk1–/–Jnk2–/– E9.0–E10.5 Forebrain

Jnk1 knockdown

Deregulated apoptosis

Hindbrain E11.5 Netrin 1

Bb Axonal commissure formation and stability

E12.5

Wild-type

AC Wild-type

Jnk1–/– at P12

CC

Wild-type

Jnk1–/–

CP

Jip3–/–

Cb Cell positioning in cortical layers

Ca Multipolar-transit and radial migration

Jnk1–/–

MZ

CP t < 10 h

IZ

Layers II–VI

t > 10 h

Floor plate

Floor plate

Exencephaly

IZ

VZ

VZ

SP CC Neuronal progenitor

Multipolar cell

D Dendrite architecture regulation Da Cerebellum Wild-type

Jnk1–/– or JBD+

Bipolar cell

Cajal–Retzius cell

Db Cortex

MKK7–JNK

CP cell

Control

SP cell

Taok2 knockdown

Dendrite (Axons not shown)

Figure 4 | JNK function in brain development.  JUN amino-terminal kinases (JNKs) are functional in various stages of brain development, as shown by various knockout and knockdown approaches, which leadNature to various development Reviews | Neuroscience defects. A | At embryonic day 9 (E9.0), mice in which both Jnk1 and Jnk2 are knocked out (Jnk1−/−Jnk2−/− mice) show reduced death in the hindbrain16,72. By E10.5, there is dramatically increased death in the forebrain16,72. By E11.5, Jnk1−/−Jnk2−/− mice show conspicuous hindbrain exencephaly, displaying open neural tubes, and by E12.5 they display protrusion of an enlarged and morphologically abnormal brain. Outside the nervous system, development seems to be normal16,72. Ba | Netrin 1, acting via the Down’s syndrome cell adhesion molecule (DSCAM) receptor (not shown), is a chemoattractant that activates JNK in the developing nervous system92. This activation is required for midline crossing in the spinal cord. Short hairpin RNA (shRNA) knockdown of Jnk1 prevents midline crossing at the floor plate92. Bb | In mice lacking the JNK scaffold JNK-interacting protein 3 (Jip3–/– mice) (which show reduced levels of basal JNK activity), the axon tracts of the corpus callosum (CC) and the anterior commissure (AC) form; however, in both cases, they fail to cross the midline96. In Jnk1–/– mice, the AC forms but then degenerates and is lost by postnatal day 12 (P12)), indicating that physiologically active JNK is important for stabilizing this commissure26. Ca | Following their final cell division, neuronal progenitor cells undertake a multipolar transition in which they move in random directions in the intermediate zone (IZ) of the developing cortex. After this stage, which lasts over 10 hours, they assume a bipolar form and start to migrate unidirectionally towards the outer surface of the cortex, bypassing the earlier-formed layers. In Jnk1−/− mice, cells exit the multipolar phase more quickly and the subsequent bipolar cell movement occurs at an accelerated rate30. Accelerated radial migration is also observed upon expression of JNK-binding domain (JBD)76. Cb | Cellular organization in the cortical layers is disturbed in Jnk1−/− mice: neuronal cells are malpositioned compared with those in wild-type mice and the subplate (SP) is less well defined30. This may be the result of faster multipolar phase transition and accelerated bipolar cell movement in Jnk1−/− mice. Analogous malpositioning of neurons is observed in the cerebellum upon targeted deletion of Mkk4, the upstream activator of JNK185. Da | Dendritic architecture is altered upon interference with JNK signalling. In the cerebellum, dendrite architecture is more complex (owing to an increased number of primary dendrites) in cerebellar granule neurons isolated from Jnk1−/− mice or in cerebellar granule neurons expressing the JBD inhibitor of JNK, whereas arbor length is reduced7. Db | In the developing cortex, JNK is activated by semaphorin 3A, which signals via neuropilin 1 to the upstream regulator of the JNK cascade, thousand-and-one amino acid kinase 2 (TAOK2). Knockdown of Taok2 with shRNA decreases JNK1 activity and reduces basal dendrite complexity in cortical neurons, whereas overexpression of a constitutively active JNK chimaera (MKK7–JNK) increases basal dendrite complexity15. Apical dendrite architecture was unchanged in both cases. CP, cortical plate; MZ, marginal zone; VZ, ventricular zone.

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REVIEWS Nuclear JNK has been shown to reside on active pro‑ moters in differentiating neurons derived from pluri‑ potent mouse embryonic stem cells, where it directly regulates the transcription of genes involved in brain morphogenesis67 (FIG.  3c). Brain development genes regulated in the Jnk1−/− brain include the atypical cad‑ herin Celsr3 and ulk‑51‑like-kinase‑1 (REF. 30), which function in axon growth and pathfinding 88,89. Thus, JNK regulates processes in the nucleus and in the cytoplasm that are probably crucial for its dominant role in the developing brain.

Commissures Bundles of nerve fibres that connect the two cerebral hemispheres.

Pial surface The outer surface of the brain that creates the boundary between the grey matter and the cerebrospinal fluid.

JNK-binding domain (JBD). JBD is a protein domain found in JUN-amino-terminal kinase (JNK)-interacting protein 1 (JIP1) that competes with JNK for substrate binding and therefore acts as a competitive, non-catalytic site inhibitor.

Radial migration Newborn glutamatergic neurons of the mammalian neocortex move long distances in synchronized cohorts to produce the six precisely arranged cortical layers. This particular type of neuronal migration is guided by radial glial scaffolds, hence the term radial migration.

Curly-tail phenotypes This describes the phenotype found in mouse in which the spinal neural tube has failed to close.

Neuronal migration and pathfinding. Following neu‑ rulation, cell proliferation and migration are activated to initiate the next phase of brain development. A large body of evidence from isolated neurons in cul‑ ture indicates that JNK activity is required for neurite growth6,9,90–92. More recent work indicates that as early as E11, JNK regulates axonal pathfinding in the spinal cord92,93. This is triggered by netrin 1, a guidance cue that activates JNK to enable midline crossing (FIG. 4Ba) through a mechanism involving cell adhesion receptors — namely, deleted in colorectal cancer 92 and Down’s syndrome cell adhesion molecule (DSCAM)92, the main mediators of netrin attraction in the nervous system94. In the cortex, JNK is activated by the secreted guidance molecule semaphorin 3A via the neuropilin 1 receptor to regulate basal dendrite architecture15,95. Moreover, studies using genetic deletion of JNK-pathway com‑ ponents report a range of axonal anomalies. For exam‑ ple, mice lacking JIP3 display axon guidance defects96. JIP3 facilitates JNK activation by recruiting upstream regulator kinases MKK4 and MKK7, and mice lacking Jip3 display reduced JNK activity in the nervous sys‑ tem45. The anterior commissures in these mice deviate from their normal path before reaching the midline at E14.5, and late-arriving callosal axons, which exhibit decreased JNK activity, fail to cross the midline 45 (FIG. 4Bb). In Jnk1 −/− mice, the anterior commissures swell and degenerate shortly after forming, although their initial development seems to be normal26 (FIG. 4Bb), and neurons lacking Jnk1, Jnk2 and Jnk3 display signs of axonal hypertrophy 97. Together, these studies pro‑ vide evidence that, in the developing nervous system, JNKs are activated by various guidance cues and that their activity is required for correct axonal trajectories to be formed and maintained15,26,92,95,98. Whether JNKs elicit control of pathfinding through gene regulation or by phosphorylating cytoskeletal targets is not known. It is likely that both mechanisms are important in this process. The final positioning of neurons in the developing brain reflects a complex programme of coordinated cell division and migration. In the cortex, cells move from their place of birth to their final destination, undergo‑ ing two distinct modes of migration. Radial progenitors first undergo multipolar stage movement that is char‑ acterized by migration in random directions within the intermediate zone of the developing cortex. This phase is transient, after which cells assume a bipolar morphol‑ ogy and migrate unidirectionally towards the pial surface

at a defined speed99,100. There is evidence that JNK1 reg‑ ulates cell movement during corticogenesis and affects the final positioning of neurons30,101,102 (FIG. 4Ca,Cb). An early indication that JNKs may be important regulators of neuronal migration came from stud‑ ies using the JNK inhibitor JNK-binding domain (JBD) and Jnk1−/− mice30,102. Following in utero electropora‑ tion to fluorescently label the embryonic brain with green fluorescent protein (GFP), the authors carried out time-lapse monitoring of multipolar and bipolar cell movements in Jnk1−/− tissues ex vivo. They found that in Jnk1−/− brains, the cells spent less time in the multipolar stage, and upon completion of this stage, the bipolar cells in the Jnk1−/− cortex moved faster than those from the wild-type cortex30 (FIG. 4Ca). Similarly, inhibition of JNK in the cytosol through the expres‑ sion of JBD accelerated radial migration in the develop‑ ing cortex, supporting the idea that JNK can negatively regulate the movement of neurons 102. By contrast, exogenous expression of a dominant-negative JNK in the developing cortex 101 or of a nuclear-targeted JBD30 inhibits radial migration. These distinct outcomes using different inhibitory approaches may reflect the opposing functions of nuclear and cytosolic JNK, or of JNK isoforms, in the regulation of migration. The net impact of JNK on migration in the cortex may ultimately depend on the surrounding extracellular cues. Indeed, in the presence of endothelin, a negative regulator of migration, JNK is required to impede cell movement 102. Outside the nervous system, a large body of literature concurs that JNK positively regulates cell migration73. One possible explanation for a differing outcome in neurons could be the existence of neuron-specific sub‑ strates. For example, ectopic expression of the neuronenriched JNK substrate MARCKSL1 in fibroblast cells switches the migration phenotype from one in which JNK facilitates migration to one in which JNK inhib‑ its migration85. The existence of neuron-specific JNK substrates (Supplementary information S1 (table)) may define a distinct molecular signature for JNK signalling in the brain. Upstream regulators of MAPK pathways have also been studied in the context of radial migration. Mekk4−/− mice exhibit highly penetrant neural tube defects, dis‑ playing cranial exencephaly and curly-tail phenotypes103. It was subsequently shown that radial migration was delayed in the absence of Mekk4 (REF. 104). However, an important finding from this study was that although the activity of the MAPK p38 was reduced in Mekk4−/− mice, JNK activity was unchanged103,104, indicating that MEKK4 acts upstream of the p38 MAPK pathway rather than upstream of the JNK pathway during brain devel‑ opment. Indeed, resolving the precise roles for effector kinases (p38 versus JNK) from the genetic deletion of upstream MEKKs or MKKs is difficult because of the pathway crosstalk that emanates from MKK4 signalling to both JNK and p38 (REF. 105) (FIG. 1a). The common use of non-specific small-molecule inhibitors of JNKs adds to the challenge of identifying which MAPK, JNK or p38 exerts a given function.

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REVIEWS Dendrite architecture regulation. Early analysis of JNK in the nervous system indicated that structurally distinct JNK inhibitors increased primary dendrite number in neurons isolated from the cerebellum6. In Jnk1−/− mice, dendrite complexity was increased in the molecular layer of the cerebellum, and cerebellar granule neurons isolated from these mice displayed increased primary dendrite numbers and reduced dendrite length in vitro7 (FIG. 4Da). Electron microscopy analysis of the cortex and hippocampus of Jnk1−/− mice showed that microtubule integrity was impaired in the dendritic compartment 26, and a subsequent study suggested that microtubules may already be altered in the developing neocortex of these mice30. Similarly, in cerebellar neurons, targeted ablation of all three JNK genes altered dendrite architecture106. Although the precise mechanism is not defined, JNK is thought to mediate its effect on dendrite morphology by phosphorylating its dendrite-specific target MAP2 and, possibly, MAP1B, as the phosphorylation of both of these substrates is reduced in the Jnk1−/− brain7,26,27 (Supplementary information S1 (table)). Extrinsic factors that activate JNK1 to control dendrite shape are starting to be identified. The secreted guidance cue semaphorin 3A is one such factor. It acts via the neu‑ ropilin 1 receptor, which interacts with thousand-andone amino acid kinase 2 (TAOK2) to activate JNK and increase basal dendrite complexity in neurons isolated from the cortex 15 (FIG. 4Db). Receptor-mediated activation of JNK also occurs via the bone morphogenetic protein receptor II (BMPRII) with which JNK1 directly inter‑ acts27. BMPRII controls dendrite modelling in many neu‑ ron types27. In cortical neurons, BMPRII activates JNK1 to stabilize microtubules and increase primary dendrite number 27, whereas in the hippocampus, JNK is activated by the non-canonical WNT pathway to increase dendrite complexity 98. The final consequence of JNK inhibition, increased or decreased dendrite complexity, thus seems to differ between neuronal cell types (FIG. 4D). Overall, JNK has emerged as a kinase that affects den‑ drite structure during development; this role may at least partly explain the association of genetic disruption of the JNK cascade components with psychiatric disorders (see below). However, more questions remain; for example, does JNK affect spine structure?

Long-term depression (LTD). A reduction of synaptic strength after application of a long-term, low-intensity stimulus.

Long-term potentiation (LTP). An increase in synaptic strength after application of a strong tetanus.

JNK function in the adult brain Synaptic plasticity. Synaptic plasticity, the ability of syn‑ apses to strengthen or weaken their responses over time, is central to cognitive function. Critical steps in synaptic plasticity involve NMDA glutamate receptor (NMDAR) activation, calcium influx and recruitment of AMPARs, all of which lead to long-lasting changes in synaptic transmission. JNK is activated by NMDA107,108 and, in turn, the JIP scaffold is required for NMDAR function109. The identified substrates of JNKs include proteins that are important regulators of synaptic plasticity. JNK1 phosphorylates PSD95 on serine 295, causing its enrich‑ ment at synapses42,110, where it sequesters cell-surface AMPAR subunits and thereby enhances postsynaptic cur‑ rents42,111 (FIG. 2d). In addition to phosphorylating PSD95, JNK1 phosphorylates the long splice form of the GluR2

AMPAR subunit (GluR2L) and facilitates its insertion at the cell surface in response to NMDAR stimulation43 (FIG. 2d). Surprisingly, however, it is long-term depression (LTD) that is absent in Jnk1−/− mice and in rats treated with the JNK inhibitor SP600125 (REF. 112); long-term potentiation (LTP) of synaptic current is unchanged in these animals112. Nonetheless, loss of LTD in Jnk1−/− mice may involve PSD95, as mutational inactivation of PSD95 eliminates LTD110,113. Alternatively, loss of JNK1 regulation of phosphatase expression may be involved, as JNK occu‑ pies the active promoter of the protein phosphatase 2A (PP2A) regulator subunit PPP2R1A67 (FIG. 3c) and PP2A is required during LTD114. However, Jnk1−/− mice112 and JNK inhibitor-treated rats110 exhibit reduced basal synap‑ tic transmission, so decreased neurotransmitter release may also contribute to the absence of LTD. Interestingly, JNK2 is required for late-phase LTP, a mechanism that underlies memory formation22, and for LTP in stressed mice115. The mechanism of JNK2 action in late-phase LTP is not known, but PSD95 and GluR2L may be involved, as they have not been ruled out as targets for JNK2‑mediated phosphorylation. Indeed, other, yet‑to‑be‑defined JNK targets presumably also contribute to plasticity changes. Protein transport. Long-range cargo transport is essen‑ tial for neuronal function. Studies in D. melanogaster were the first to highlight a role for JNKs as regulators of motor transport in neurons. In flies, the Basket scaffold Sunday driver (the fly homologue of JIP3) was identi‑ fied as an adaptor protein that tethered axonal cargo to kinesin light chain116, and later, JIP1 was found to be a cargo adaptor in mammalian cells117. Around the same time, in Caenorhabditis elegans, components of the JNK pathway — UNC‑16 (the worm JIP3 homologue) as well as JNK and MKK4 homologues — were shown to be nec‑ essary for normal protein transport118. In hippocampal neurons, JIP1 is transported from the neurites to the peri‑ nuclear region after oxygen glucose deprivation stress. This may contribute to the death mechanism, as neu‑ rons lacking Jip1 are protected from this treatment 119,120. In peripheral axons, JNK3, kinesin 1 and JIP3 exist in a complex 23. Following axotomy, JNK3 is activated and anterograde transport is disrupted, whereas retrograde transport increases23, suggesting that JNK3 acts as a sur‑ veillance sensor that responds to stress by altering motor transport. Consistent with this indication, mutant hun‑ tingtin (mHTT) has been reported to inhibit fast axonal transport through a mechanism involving JNK3, which has implications for Huntington’s disease121 (see below) (FIG. 5a). JNK is also implicated in regulation of kinesin 1 function under physiological conditions in mammalian neurons, as JNK phosphorylation of JIP1 has been shown to control the directionality of amyloid precursor protein (APP) transport in axons120.

Role in disease JNKs can elicit neuronal death in a wide range of patho‑ logical contexts, as shown by the numerous reports docu‑ menting broad neuroprotection upon treatment with JNK inhibitors or genetic deletion of JNK52,122. Early studies using Jnk3−/− mice highlighted JNK3 as a main isoform

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REVIEWS a Huntington’s disease

protects neurons from NMDA-induced excitotoxicity in vitro 25,108,132, from cerebral ischaemia108,127,133–135 and from kainate-induced epilepsy in which gliosis is also reduced136,137. Even ischaemic preconditioning has been attributed to downregulation of JNK signalling 138,139. Consequently, JNK has emerged as a central mediator of excitotoxic damage in the nervous system.

Cargo Axon transport

mHTT

P

P

P

JNK3

Axon transport

MT

Kinesin 1

Axon

b Axotomy

Degraded SCG10

SCG10 P

Axonal degeneration

P

P

JNK

P JNK

P

Axon fragmentation

P Proximal

Distal

Figure 5 | JNK regulates stress responses in the axon.  JUN Reviews amino-terminal kinase Nature | Neuroscience (JNK) has been found to regulate protein transport and protein homeostasis in models of Huntington’s disease and Wallerian degeneration. a | Mutant huntingtin (mHTT) — which causes Huntingtons’ disease — activates JNK3 (REF. 158) and, in turn, JNK3 phosphorylates the kinesin 1 motor domain, which leads to dissociation of kinesin 1 from microtubules (MTs) and impaired axonal transport121. b | Regulated protein degradation is thought to drive Wallerian degeneration of axons in response to injury. In healthy neurons, superior cervical ganglion 10 protein (SCG10) homeostasis is maintained through a balance of continuous degradation, requiring JNK activity, and transport-mediated replenishing of pools. After axonal injury in peripheral neurons, JNK phosphorylation of SCG10 facilitates its degradation in the distal region of the axon. Injury of peripheral nerves blocks transport and delivery of SCG10 distal to the injury site, leading to degeneration125.

triggering neuronal death, including in response to expo‑ sure to excitotoxic insults52, amyloid-β123, 6‑hydroxydo‑ pamine19 or axotomy 19,124; JNK3 is also implicated in Huntington’s disease121. Other JNK isoforms may also contribute to neuronal death. Indeed, neurons isolated from Jnk3−/− or Jnk2−/−Jnk3−/− mice are not protected from trophic withdrawal-induced cell death, whereas small interfering RNA knockdown of all three Jnk isoforms imparts notable neuroprotection28. Similarly, in the per‑ manent occlusion ischaemia model, genetic deletion of Jnk2 and Jnk3 is required to reduce damage in the cortex of mice19. Furthermore, JNK is implicated in the axonal degeneration pathway, in which it drives the breakdown of SCG10, which is rapidly lost from the axon after axonal injury 125 (FIG. 5b). Interestingly, pan-JNK inhibitors have shown efficacy in various disease models126, and spatial targeting of JNKs has shown promise as a neuroprotective approach28,127.

Ischaemic preconditioning This term refers to the protection rendered by exposure to sequential periods of sublethal ischaemia.

Stroke and epilepsy. Excitotoxic death, a consequence of excessive glutamate receptor stimulation, occurs in stroke, status epilepticus, traumatic brain injury and in various neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease128,129. JNKs are activated by glutamate, the most prevalent excitatory neurotransmitter in the nervous system130, through NMDARs and AMPARs42,43,107,131. In a landmark study, Jnk3−/− mice were found to be resistant to kainate-induced seizures 52. Subsequent studies reported that pharmacological inhibition of JNK

Alzheimer’s disease. Alzheimer’s disease is a progressive degenerative disorder of the CNS that is characterized by amyloid‑β deposits (plaques) and neurofibrillary tangles140. Post-mortem brains from patients with this disease have been shown to display anomalously high levels of JNK activity 141–143, and preclinical studies using animal models indicate that JNK may markedly affect Alzheimer’s disease pathology by increasing amyloid‑β plaque load106,143 and by hyperphosphorylating tau129,144,145. In mouse models of Alzheimer’s disease in which animals express the Swedish mutant variant of APP, from which amyloid‑β is derived, and/or harbour a mutation in pre‑ senilin 1 (which is involved in APP cleavage), JNK activ‑ ity is increased143,146,147. Application of JNK inhibitors to brain slices from one such model (the Tg2576/PS1P264L mouse) in vitro reduces degeneration of pyramidal neu‑ rons148, and chronic administration of a peptide JNK inhibitor to TgCRND8 mice (carrying several mutations in App) rescues memory impairment and LTP defects149. Moreover, genetic deletion of Jnk3 in familial Alzheimer’s disease mice decreases amyloid‑β plaque load143. JNK phosphorylates APP on threonine 668 (REFS 106,150), a critical event for amyloidogenic processing 151. Consistent with this, disruption of both Mkk4 and Mkk7, which decreases JNK activity in the cortex by 70%106, reduces amyloid‑β plaque load in TgCRND8 mice, as does chronic JNK inhibitor treatment 149. Alongside glycogen synthase kinase 3 (GSK3), p38 and ERK, JNK phospho‑ rylates tau on multiple sites that are hyperphosphorylated in paired helical fragments144,145,152. Increased JNK activ‑ ity has been detected in neurofibrillary tangles in brain tissue from patients with Alzheimer’s disease153. Also, in the Tg2576/PS1P264L and traumatic brain injury mouse models, JNK activity is increased in tangles, where JNK localizes with phosphorylated tau146,154. Of note, a peptide inhibitor of JNK, D-JNKI‑1, reduces tau phosphorylation and aggregation154. Parkinson’s disease. Various lines of evidence support a role for JNK in Parkinson’s disease. Of note, JNK activity is increased in post-mortem brain tissue from patients with this disorder 141,142. A breakthrough early finding was that the adenosine triphosphate (ATP)-competitive inhibi‑ tor of MLK CEP‑1347 conferred neuroprotection in the MPTP (1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridin) model of Parkinson’s disease155, and several subsequent studies reported anti-apoptotic actions of CEP‑1347 in cell and animal models of this disease156. CEP‑1347 entered a clinical trial for the treatment of Parkinson’s disease. However, the trial was eventually discontinued, and the results were inconclusive; it was not established whether an effective dose for inhibition of JNK was achieved157. Indeed, the outcome of this trial emphasizes the need

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REVIEWS for a biomarker readout of efficacy for Parkinson’s dis‑ ease and possibly highlights the shortcomings of exist‑ ing preclinical models of Parkinson’s disease for drug development157. Huntington’s disease. Huntington’s disease is caused by HTT that has a polyglutamine expansion (mHTT). JNK is activated by mHTT, and dominant-negative inhibition of JNK protects from mHTT-mediated toxicity 158. The disease mechanism is associated with JNK3‑dependent phosphorylation of kinesin 1, leading to its dissociation from microtubules and inhibition of fast axonal transport 121 (FIG. 5a). Psychiatric diseases. Anomalies in dendrite and synapse structure are a consistent hallmark of neuropsychiatric disorders. New evidence from human genetics studies identified genes associated with the JNK pathway that seem to confer susceptibility to autism spectrum disor‑ ders, schizophrenia and intellectual disability 10,12–14. The genetic basis for these heritable conditions is mostly unknown, making the findings of particular interest. Directly upstream of JNK, a genetic association study identified two single-nucleotide polymorphisms in MKK7 that confer genetic risk for schizophrenia, sug‑ gesting that reduced function of JNK may underlie the neurochemical changes and core symptoms of this disor‑ der 12. Interestingly, one of the polymorphisms was in the 3′ untranslated region of MKK7 (REF. 12), which targets MKK7 mRNA to growth cones24. This gene is thought to have escaped earlier detection with genome-wide asso‑ ciation study approaches because of poor representation on the arrays used in these studies. In humans, TAOK2 is located on chromosome 16p11.2, a region that carries substantial susceptibility to autism spectrum disorders14 and schizophrenia159. TAOK2 was recently shown to account partially for acti‑ vation of JNK1 in the brain and to regulate basal dendrite formation in the cortex of mice15. An additional sign that JNK may have a central role in these disorders comes from study of the gene encoding interleukin‑1 receptor accessory protein-like 1 (IL1RAPL1)160, which is impli‑ cated in monogenic forms of mental retardation and autism. IL1RAPL1 signals through JNK, and mice null for Il1rapl1 show reduced JNK activity 160. Finally, gene anomalies in the effector kinase itself have been identified. Chromosomal translocation leading to a loss-of-function truncation of JNK3 has been identified in cohort and case studies of individuals with intellectual disabilities10,11,13.

JNKs as therapeutic targets Targeting JNKs in neurons. The pursuit of orally active JNK inhibitors for the treatment of nervous system disor‑ ders (cerebral ischaemia, sound trauma, multiple sclerosis and neurodegenerative diseases) has spanned a period of over 10 years. Such inhibitors must be efficiently adsorbed, penetrate the blood–brain barrier and be well tolerated. Several classes of small-molecule, ATP-competitive inhibi‑ tors of JNKs have been identified by screening chemical libraries for inhibitors of kinase activity using isotopic labelling, antibody-based assays (ELISAs), time-resolved

fluorescence or fluorescence anisotropy measurements132. The first reported small-molecule JNK inhibitor was SP600125, an anthrapyrazalone that inhibits JNK1, JNK2 and JNK3 (REF. 161) (inhibition constant is ~0.19 μM). SP600125 is neuroprotective in animal models of stroke, even when administered 1 hour after the initiation of global transient ischaemia162, and improves functional recovery after spinal cord injury in mice163. However, although SP600125 is commonly used as a JNK inhibi‑ tor in preclinical studies, it is not specific164 — it inhibits 74 kinases (from 353 identified from the KINOMEscan Library of Integrated Network-based Cellular Signatures database) at 10 μM. Indeed, delayed treatment with SP600125 at 7 days after occlusion worsens the out‑ come in a rat model of transient focal cerebral ischae‑ mia, increasing infarct volume and inhibiting vascular remodelling 165. Initial JNK inhibitors showed poor bioavailability and had to be administered by intraventricular injections or infusions to achieve effective doses126. Compounds with improved oral bioavailability and CNS penetration have been developed. These thiophene-based JNK inhibitors have increased potency (in vitro median inhibitory con‑ centration (IC50) of 0.01–0.04 nM in the presence of 10 μM ATP), improved pharmacokinetic properties and reduced neurotoxicity in an amyloid‑β neurotoxicity assay 166. The idea that improved neuroprotection could be achieved through selective inhibition of JNK isoforms drove the search for isoform-specific inhibitors, par‑ ticularly for JNK2 and JNK3 isoforms, which exhibit clear pro-death properties19,21. The developed inhibitors included those that showed more than tenfold selectiv‑ ity for JNK2 and JNK3 over JNK1 (REF. 167) and thiazole compounds that inhibited JNK3 and JNK1 above JNK2 (REF. 168). However, these inhibitors all showed poor CNS penetration in vivo168 despite showing neuroprotection in vitro 167. JNK3‑specific aminopyrimidine inhibitors with good CNS permeability and bioavailability were developed, although they also inhibited cytochrome P450, which is important for lipid, hormone and drug metabolism. Thus, they were considered to be non-via‑ ble as therapeutics because of anticipated adverse side effects169. Recently, structurally similar pyridine-based compounds displaying high potency against JNK3 and good bioavailability were described170, and 1‑aryl‑1H‑in‑ dazole molecules showing 10–20‑fold specificity for JNK3 above JNK1 have also been developed, some of which show improved CNS penetration and pharmacoki‑ netics171. Despite the effort to develop isoform-specific JNK inhibitors for CNS disorders, JNK inhibitors that do not distinguish well between JNK isoforms demonstrate the most neuroprotection in vivo126, consistent with the proposal that all JNKs contribute to neuronal death28. In addition to small-molecule JNK inhibitors, a pep‑ tide inhibitor of JNKs (D‑JNKI‑1) has been developed and extensively studied. D‑JNKI‑1 exploits a Tat delivery sequence from HIV to cross cell membranes and com‑ prises D‑amino acids to confer greater stability against proteolytic cleavage172. D‑JNKI‑1 includes a 20 aminoacid sequence from JIP1. Initial work demonstrated that D‑JNKI‑1 offered remarkable neuroprotection in

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REVIEWS rodent models of ischaemia and inflammation108,132. Intraventricular administration of D‑JNKI‑1 at 11 mg per kg, as late as 6 hours after occlusion, reduced infarct vol‑ ume in the mouse middle cerebral artery occlusion model. The effects lasted for at least 14 days108. Intravenous injec‑ tion of a single dose of D‑JNKI‑1 several hours after ischaemia was also protective in this stroke model and was compatible with the presence of tissue plasminogen activator, suggesting that D‑JNKI‑1 may be a promising agent for the treatment of stroke173. Inhibition of JNK in particular locations. Compartmentspecific targeting of JNK has been used to selec‑ tively block pro-apoptotic JNK action in the nucleus, in the cytoplasm and at mitochondria (TABLE 2; see Supplementary information S2 (figure)). Targeted inhibitors of JNK have been generated by fusing a nuclear export sequence (NES) or a nuclear localization sequence (NLS) upstream of the JNK inhibitor JBD, resulting in constructs with demonstrated selectivity of action in the cytosol and nucleus, respectively 7,9. Thus, in mice, expression of GFP–NES–JBD mimics the knockout

of Jnk1 in that it causes acceleration of transit in the multipolar stage and radial migration in the developing brain30, a reduction in neurite growth10 and an increase in dendrite complexity 7. By contrast, inhibition of JNK in the nucleus upon expression of GFP–NLS–JBD does not disrupt these physiological events7,9,30. Interestingly, however, GFP–NLS–JBD provides substantial protec‑ tion from neuronal death in response to withdrawal of trophic support 28 and death evoked by activation of the p75 neurotrophin receptor 174 (FIG. 5). This suggests that the nucleus is a major compartment in which JNK elicits death responses, and gene regulation would seem the probable mechanism by which it acts. In support of this, directing JNK activators to the nucleus increases neu‑ ronal toxicity, and structurally distinct nuclear inhibitors protect against toxicity 28. It is notable that a fluorescently labelled Tat–D-JNKI‑1 peptide inhibitor of JNK accu‑ mulates passively in the nuclei of neurons in the cortex within 1 hour of being intraperitoneally injected into rats175. This inhibitor provides protection against exci‑ totoxic damage in cultured neurons and in mouse and rat models of cerebral ischaemia25,108,127.

Table 2 | JNK-based therapeutic approaches Agent

Type

Physiological effects

Effects after death stimulus

NES–JBD

Cytosolic JNK inhibitor

• Accelerated multipolar transition30 • Accelerated radial migration30 • Altered dendrite architecture7

• Does not protect against p75NTR‑induced death174 • Does not inhibit caspase 3 activation in response to p75NTR stimulation174 • Does not protect against trophic withdrawal-induced death28

JBD

JNK inhibitor with inherent cytosolic localization

• Accelerated radial migration102 • Altered dendrite architecture7

Not determined

NLS–JBD

Nuclear JNK inhibitor

• Retarded radial migration30 • No alteration of dendrite shape7

• Inhibits caspase 3 activation upon p75NTR stimulation174 • Protects against p75NTR‑mediated death174 • Protects against trophic deprivationinduced death28

NES–MKK4‑kd Cytosolic dominant-negative inhibitor of JNK

Not determined

Does not protect against trophic deprivationinduced death28

NLS–MKK4‑kd Nuclear dominant-negative inhibitor of JNK

Not determined

Protects neurons from trophic deprivationinduced death28

D-JNKI‑1 (XG‑102)

Cell-permeable peptide inhibitor of JNK172

• Reduced basal synaptic transmission at higher doses110 • Impaired long-term depression110

• Protects neurons from NMDA-induced death25 • Reduces damage after cerebral ischaemia in the mouse and rat108,127,134 • Rescues memory impairment and reduces amyloid load in the TgCRND8 AD mouse149 • Reduces tau phosphorylation and aggregation154

Tat-SAB–KIM1

Cell-permeable peptide that blocks JNK association with mitochondria176

Not determined

Reduces damage after cerebral ischaemia in the postnatal day 7 rat127

SP600125

ATP-competitive inhibitor of JNK161

Inhibits long-term depression112

Protects against cerebral ischaemia162 and spinal cord injury163

AD, Alzheimer’s disease; ATP, adenosine triphosphate; JBD, JNK-binding domain; JNK, JUN amino-terminal kinase; KIM1, kinase interaction motif 1; MKK4‑kd, MKK4‑kinase dead; NES, nuclear export sequence; NLS, nuclear localization sequence; p75NTR, p75 neurotrophin receptor.

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VOLUME 15 | MAY 2014 | 295 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Specific inhibition of JNK at mitochondria has also been another successful targeting approach. JNK is found enriched at mitochondria following excitotoxic stresses, transient middle cerebral artery occlusion and treatment with kainate34,35. A peptide interfering with JNK recruitment to mitochondria has been developed. The cell-permeable peptide corresponding to the kinase interaction motif 1 (KIM1) domain of the mitochdon‑ drial JNK scaffold SAB (also known as SH3BP5)176 reduces damage in a rat model of neonatal ischaemia127. In HeLa cells, SAB–KIM1 prevents BCL‑2 and BCL-XL phosphorylation176, JNK-dependent modifications that antagonize the protective functions of BCL‑2 and BCL-XL at mitochondria. However, it is worth noting that although the death function of these proteins is exe‑ cuted in the cytoplasm, the initial trigger for the rapid induction of BH3‑only proteins occurs in the nucleus and is dependent on JNK38. Thus, it seems reasonable that targeting of JNK either in the nucleus or at mito‑ chondria may confer protection. Peptide drugs provide the possibility for organelle targeting. Conveniently, in the case of nuclear targeting, the HIV-derived Tat Kyriakis, J. M. & Avruch, J. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10‑year update. Physiol. Rev. 92, 689–737 (2012). 2. Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010). 3. Lundby, A. et al. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. Nature Commun. 3, 876 (2012). 4. Coffey, E. T. & Courtney, M. J. Regulation of SAPKs in CNS neurons. Biochem. Soc. Trans. 25, S568 (1997). 5. Hu, Y., Metzler, B. & Xu, Q. Discordant activation of stress-activated protein kinases or c‑Jun NH2‑terminal protein kinases in tissues of heat-stressed mice. J. Biol. Chem. 272, 9113–9119 (1997). 6. Coffey, E. T., Hongisto, V., Dickens, M., Davis, R. J. & Courtney, M. J. Dual roles for c‑Jun N‑terminal kinase in developmental and stress responses in cerebellar granule neurons. J. Neurosci. 20, 7602–7613 (2000). 7. Björkblom, B. et al. Constitutively active cytoplasmic c‑Jun N‑terminal kinase 1 is a dominant regulator of dendritic architecture: role of microtubule-associated protein 2 as an effector. J. Neurosci. 25, 6350–6361 (2005). This study shows that dendrite architecture is disturbed in cerebellar granule neurons isolated from Jnk1–/– mice. 8. Kuan, C. Y. et al. A critical role of neural-specific JNK3 for ischemic apoptosis. Proc. Natl Acad. Sci. USA 100, 15184–15189 (2003). 9. Tararuk, T. et al. JNK1 phosphorylation of SCG10 determines microtubule dynamics and axodendritic length. J. Cell Biol. 173, 265–277 (2006). 10. Shoichet, S. A. et al. Truncation of the CNS-expressed JNK3 in a patient with a severe developmental epileptic encephalopathy. Hum. Genet. 118, 559–567 (2006). A study providing the first evidence of a JNK gene disruption in a patient with learning disability and epilepsy. This link has been supported by subsequent case studies. 11. Baptista, J. et al. Breakpoint mapping and array CGH in translocations: comparison of a phenotypically normal and an abnormal cohort. Am. J. Hum. Genet. 82, 927–936 (2008). 12. Winchester, C. L. et al. Converging evidence that sequence variations in the novel candidate gene MAP2K7 (MKK7) are functionally associated with schizophrenia. Hum. Mol. Genet. 21, 4910–4921 (2012). The article provides the first genetic link between the JNK pathway and schizophrenia. This human cohort study reports the association of MKK7 anomalies with schizophrenia. 1.

sequence targets passively to nuclei and nucleoli175,177. It therefore seems obvious that delivering a drug directly to its site of action could be a favourable therapeutic strategy.

Conclusions JNK emerges from these studies as a key player in the developing and adult brain. Interruption of JNK signal‑ ling alters neuronal pathfinding, migration and axoden‑ dritic architecture as well as synaptic function. Genetic interference at several levels of the JNK pathway confers susceptibility to psychiatric disease. However, a complete picture of how JNKs coordinate these events is lacking, as many new substrates remain to be identified. If one-third of brain proteins are phosphorylated on proline-directed residues2, the number of genuine targets for the JNK fam‑ ily of kinases will probably be extensive. These will com‑ prise both gene regulators and extra-nuclear targets that reside in various neuronal subcompartments. Indeed, spatially targeted inhibitors of JNK function may pro‑ vide a strategy for neuroprotection with fewer unwanted adverse effects.

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Acknowledgements

The author thanks D. Flinkman and P. James for critically reading the manuscript. This research was supported by Åbo Akademi University and the Academy of Finland.

Competing interests statement

The author declares no competing interests.

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Nuclear and cytosolic JNK signalling in neurons.

It has been over 20 years since JUN amino-terminal kinases (JNKs) were identified as protein kinases that are strongly activated by cellular stress an...
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