ZPK/DLK, a mitogen-activated protein kinase kinase kinase, is a critical mediator of programmed cell death of motoneurons - PubMed (original) (raw)

Comparative Study

ZPK/DLK, a mitogen-activated protein kinase kinase kinase, is a critical mediator of programmed cell death of motoneurons

Aki Itoh et al. J Neurosci. 2011.

Abstract

Activation of mitogen-activated protein kinase pathways is critically involved in naturally occurring programmed cell death of motoneurons during development, but the upstream mediators remain undetermined. We found that mice deficient in ZPK, also called DLK (ZPK/DLK), an upstream kinase in these pathways, have twice as many spinal motoneurons as do their wild-type littermates. Nuclear HB9/MNX1-positive motoneuron pools were generated similarly in the spinal cord of both ZPK/DLK-deficient and wild-type embryos. Thereafter, however, significantly less apoptotic motoneurons were found in ZPK/DLK-deficient embryos compared with wild-type embryos, resulting in retention of excess numbers of motoneurons after birth. Notably, these excess motoneurons remained viable without atrophic changes in the ZPK/DLK-deficient mice surviving into adulthood. Analysis of the diaphragm and the phrenic nerve revealed that clustering and innervation of neuromuscular junctions were indistinguishable between ZPK/DLK-deficient and wild-type mice, whereas the proximal portion of the phrenic nerve of ZPK/DLK-deficient mice contained significantly more axons than the distal portion. This result supports the hypothesis that some excess ZPK/DLK-deficient motoneurons survived without atrophy despite failure to establish axonal contact with their targets. This study provides compelling evidence for a critical role for ZPK/DLK in naturally occurring programmed cell death of motoneurons and suggests that ZPK/DLK could become a strategic therapeutic target in motor neuron diseases in which aberrant activation of the apoptogenic cascade is involved.

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Figures

Figure 1

ZPK/DLK-deficient mice have more than twice as many spinal motoneurons as do wild-type mice. A. Six μm-thick cross-sections of the L4 (fourth lumbar) spinal segments from wild-type (+/+) and ZPK/DLK-deficient (tp/tp) mice at postnatal day 21 (PN21) were immunolabeled for ChAT (red) and neuron-specific nuclear protein (NeuN, green). Motoneurons were identified as a ChAT+ neuronal subset (yellow to red). B. Quantitative analysis of motoneurons in the C4 (fourth cervical), Th4 (fourth thoracic), and L4 spinal segments of wild-type and ZPK/DLK-deficient mice at PN21 and in adulthood. Motoneurons with visible nucleoli were counted in at least 9 sections per animal, and the average number of motoneurons per section of the anterior horn on one side was calculated. At least three animals per genotype were examined in each age group. C. Axons in the L4 anterior root were also increased in number in ZPK/DLK-deficient mice. Representative confocal images of NF200-positive axons (inverted) in the L4 anterior roots from wild-type and ZPK/DLK-deficient mice are shown. D. Quantitative analysis of axon numbers in the L4 anterior roots (AR) from PN21 and adult mice as in B. E. A representative set of frequency histograms revealed that axons were increased in the entire range of axon sizes in ZPK/DLK-deficient mice at PN21. F. Motoneuron NPCD was reduced in ZPK/DLK-deficient mice. The lumbar spinal segments from wild-type (+/+) and ZPK/DLK-deficient (tp/tp) embryos at E12.5 and E13.5 were immunolabeled for HB9 (green) and cleaved Caspase-3 (red). Nuclei were counterstained with DAPI (blue). The clusters of HB9-positive cells (in the areas outlined by rectangles in the left column) were enlarged in the right column. At E13.5, cleaved Caspase-3-positive cells were frequently observed in the HB9-positive motoneuron cluster of the wild-type spinal cord (arrows). These cells had pyknotic nuclei typical of cells undergoing apoptosis. Arrowheads indicate apoptotic cells outside of the motoneuron clusters. ** indicates p<0.01 (unpaired _t_-test). Scale bars; A, C, and F, 100 μm.

Figure 2

Innervation of the diaphragm by the phrenic nerve was indistinguishable between ZPK/DLK-deficient and wild-type mice. Whole-mount diaphragms and distal phrenic nerves from wild-type (+/+) and ZPK/DLK-deficient (tp/tp) embryos at E16.5 were immunolabeled for NF200 (green) and α-bungarotoxin (α-Bgtx, red). The portions pointed by the arrows in panels A and B are magnified in panels C and D, respectively, to demonstrate terminal branching of the phrenic nerve and neuromuscular junctions. E and F, Representative Z-stacked confocal images of the α-Bgtx labeled neuromuscular junctions from the bottom to top surfaces of the diaphragm from wild-type (E) and ZPK/DLK-deficient (F) E16.5 embryos demonstrate no obvious increase in clustering of neuromuscular junctions. At PN21, all of the neuromuscular junctions examined were innervated by a single axon in the diaphragms of both ZPK/DLK-deficient (H) and wild-type (G) mice. Representative pictures are shown. Scale bars; A, 500 μm (for A and B); C and E, 100 μm (for C-F); and G, 10 μm (for G and H).

Figure 3

ZPK/DLK-deficient mice had more myelinated axons in the proximal portion of the phrenic nerve than in the distal portion. A–D, Representative confocal laser micrographs of proximal (A and B) and distal (C and D) portions of the wild-type (+/+) and ZPK/DLK-deficient (tp/tp) phrenic nerves immunolabeled for MBP and NF200. The proximal portion was at the level of the aortic arch, and the distal portion was immediately proximal of the first major trifurcation of the phrenic nerve adjacent to the diaphragm. E–H, Methylene blue-stained 1 μm-thick cross sections of the proximal portions (E and F) and the distal portions (G and H) of the phrenic nerves from wild-type (E and G) and ZPK/DLK-deficient (F and H) mice. Representative high-power images demonstrate that the distal portion of the phrenic nerve from a ZPK/DLK-deficient mouse was primarily composed of large-diameter myelinated axons, which were indistinguishable from that from a wild-type mouse. I. Quantitative analysis of myelinated axon numbers in the phrenic nerves from ZPK/DLK-deficient (closed circles, tp/tp) and wild-type (open circles, +/+) mice at PN21. Each pair of the connected circles represents the myelinated axon numbers at the proximal and distal portions of a phrenic nerve. Left and right phrenic nerves from three mice were quantified in each genotype. Scale bars; D and H, 100 μm.

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ZPK/DLK, a mitogen-activated protein kinase kinase kinase, is a critical mediator of programmed cell death of motoneurons - PubMed (original) (raw)