Targeted Deletion of the Mitogen-Activated Protein Kinase Kinase 4 Gene in the Nervous System Causes Severe Brain Developmental Defects and Premature Death

Abstract

The c-Jun NH 2 -terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.The c-Jun NH 2 -terminal protein kinase (JNK) (also called stress-activated protein kinase) is a member of the mitogenactivated protein kinase (MAPK) family, implicated in the regulation of numerous cellular functions in response to environmental stresses, growth factors, hormones, and proinflammatory cytokines (11). Three genes, jnk1, jnk2, and jnk3, encoding 10 isoforms, have been cloned. JNK1 and JNK2 are ubiquitously expressed, while JNK3 is almost exclusively found in the central nervous system (CNS). To understand the physiological functions of the JNK isoforms in vivo, transgenic mice deficient in one or more of the jnk genes have been studied (11). Their phenotypic analysis highlighted the importance of JNK-mediated apoptosis during the development of the CNS and in response to brain injury (26, 43, 52). However, apoptosis does not represent the only functional consequence of JNK activation. For example, JNK1 appears to contribute to establishing dendritic architecture in the brain (3,8,42).Analogous to other MAPKs, JNK is activated via the sequential activation of protein kinases, which include two dualspecificity MAPK kinases (MKK4 and MKK7) and multiple MAPK kinase kinases (MEKKs) (51). The MEKKs phosphorylate and activate MKK4 and MKK7, which, in turn activate JNK by dual phosphorylation on Thr and Tyr residues within a Thr-Pro-Tyr motif in protein kinase subdomain VIII (51). While MKK7 is a specific activator of JNK, MKK4 can also phosphorylate the Thr-Gly-Tyr motif of the p38 MAPK (50).Like JNK, p38 MAPK is activated in mammalian cells by various stress stimuli and proinflammatory cytokines (55). Physiological evidence for a role of MKK4 in activating the p38 MAPK cascade was recently provided by demonstrating that decreased expression of MKK4 due to small interfering RNA in mouse embryonic fibroblasts lacking both MKK3 and MKK6 suppressed stress-induc...

Stress-Activated Protein Kinase MKK7 Regulates Axon Elongation in the Developing Cerebral Cortex

“…In addition, the phosphorylation of doublecortin (DCX) was suppressed by loss of MKK7 (Fig. 10C), an alteration that does not occur after disruption of MKK4 (Wang et al, 2007). These data show that, within the developing brain, MKK7 regulates the phosphoryla- tion of not only NF-H and MAP1B, which are also regulated by MKK4, but also DCX.…”

“…Last, JNK signaling is involved in neuronal migration during brain development. Cortical neuronal migration was retarded by overexpression of a dominant-negative form of JNK (Kawauchi et al, 2003), and specific deletion of MKK4 in the CNS caused a delay in neuronal radial migration in the cerebral cortex as well as misalignment of Purkinje cells in the cerebellum (Wang et al, 2007). Together, these reports demonstrate that the proper control of JNK signaling is required for normal brain development; however, the specific role of MKK7 in this control has yet to be defined.…”

“…Previous reports showed that overexpression of dominantnegative JNK or MKK4 disruption can delay neuronal radial migration in the cerebral cortex (Kawauchi et al, 2003;Wang et al, 2007). To determine whether MKK7 also affects this process, we performed a timed pregnancy study of BrdU incorporation in the developing cerebral cortex.…”

“…In Mkk4 flox/flox Nestin-Cre mice, the phosphorylation of certain neurofilament and MAPs is reportedly altered (Wang et al, 2007). Neurofilament heavy chain (NF-H) is a neuron-specific intermediate filament whose C-terminal tail is constitutively phosphorylated in axons by several proline-directed kinases, including JNK.…”

A Case Report of a Cerebellar Neuroblastoma in a p53 Null Mutation Mouse

“…Therefore, we consider that the present case could be diagnosed as a cerebellar neuroblastoma according to the WHO classification criteria [4]. Developing radial or Bergmann glias have shown to express nestin intermediate filament protein in addition to neuronal stem cells [11]. In addition, recent studies have shown that cultured nestin-positive cells from postnatal mouse small bowel have a potential to differentiate into neurons, glias, and smooth muscle cells suggesting that nestin expression in glial cells originate from neuronal stem cells [9].…”