Ryk controls remapping of motor cortex during functional recovery after spinal cord injury.

Limited functional recovery can be achieved through rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, in mice and rats by either conditional knockout in the motor cortex or monoclonal antibody infusion resulted in increased corticospinal axon collateral branches with presynaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and that hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to controlling the forelimb in Ryk conditional knockout mice than in controls (wild-type or heterozygotes). In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced and there was no significant functional recovery even in Ryk conditional knockout mice. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and targeted rehabilitation.

Altered expression of atypical PKC and Ryk in the spinal cord of a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive paralysis due to the selective death of motor neurons of unknown causes. Increasing evidence indicates that Wnt signaling is altered in ALS. In this study, we focused on two non-canonical Wnt signaling components, atypical PKC (aPKC) and a Wnt receptor, Ryk, in a mouse model of ALS, SOD1 (G93A). aPKC mediates Wnt signaling to regulate growth cone guidance, axon differentiation and cell survival. Ryk is a Wnt repulsive receptor that regulates axon guidance and inhibits regeneration after spinal cord injury. aPKC expression was increased in motor neurons of the lumbar spinal cord in SOD1 (G93A) mice at both early and late stages. Interestingly, aPKC was co-localized with SOD1 in motor neuron cell bodies and extracellular aggregates, and aPKC-containing extracellular aggregates increased with disease progression. Biochemical fractionation showed that aPKC protein level was increased in the detergent-insoluble protein fraction in SOD1 (G93A) mice at late stage but decreased in the detergent-soluble fraction at symptomatic stage. These results suggest that aPKC may be sequestered in SOD1 aggregates, impairing its ability to protect motor neurons from death. Ryk expression was also increased in the motor neurons and the white matter in the ventral lumbar spinal cord of mutant SOD1 mice with a peak at early stage. These observations indicate that Wnt/aPKC and Wnt/Ryk signaling are altered in SOD1 (G93A) mice, suggesting that changed Wnt signaling may contribute to neurodegeneration in ALS.

p57(KIP2) regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex.

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.

The multiple roles of the cyclin-dependent kinase inhibitory protein p57(KIP2) in cerebral cortical neurogenesis.

The members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitory proteins (CKIs), including p57(KIP2), p27(KIP1), and p21(CIP1), block the progression of the cell cycle by binding and inhibiting cyclin/CDK complexes of the G1 phase. In addition to this well-characterized function, p57(KIP2) and p27(KIP1) have been shown to participate in an increasing number of other important cellular processes including cell fate and differentiation, cell motility and migration, and cell death/survival, both in peripheral and central nervous systems. Increasing evidence over the past few years has characterized the functions of the newest CIP/KIP member p57(KIP2) in orchestrating cell proliferation, differentiation, and migration during neurogenesis. Here, we focus our discussion on the multiple roles played by p57(KIP2) during cortical development, making comparisons to p27(KIP1) as well as the INK4 family of CKIs.

The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors.

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).

Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex.

Although survival-promoting effects of insulin-like growth factor-1 (IGF-1) during neurogenesis are well characterized, mitogenic effects remain less well substantiated. Here, we characterize cell cycle regulators and signaling pathways underlying IGF-1 effects on embryonic cortical precursor proliferation in vitro and in vivo. In vitro, IGF-1 stimulated cell cycle progression and increased cell number without promoting cell survival. IGF-1 induced rapid increases in cyclin D1 and D3 protein levels at 4 h and cyclin E at 8 h. Moreover, p27(KIP1) and p57(KIP2) expression were reduced, suggesting downregulation of negative regulators contributes to mitogenesis. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway specifically underlies IGF-1 activity, because blocking this pathway, but not MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase), prevented mitogenesis. To determine whether mechanisms defined in culture relate to corticogenesis in vivo, we performed transuterine intracerebroventricular injections. Whereas blockade of endogenous factor with anti-IGF-1 antibody decreased DNA synthesis, IGF-1 injection stimulated DNA synthesis and increased the number of S-phase cells in the ventricular zone. IGF-1 treatment increased phospho-Akt fourfold at 30 min, cyclins D1 and E by 6 h, and decreased p27(KIP1) and p57(KIP2) expression. Moreover, blockade of the PI3K/Akt pathway in vivo decreased DNA synthesis and cyclin E, increased p27(KIP1) and p57(KIP2) expression, and prevented IGF-1-induced cyclin E mRNA upregulation. Finally, IGF-1 injection in embryos increased postnatal day 10 brain DNA content by 28%, suggesting a role for IGF-1 in brain growth control. These results demonstrate a mitogenic role for IGF-1 that tightly controls both positive and negative cell cycle regulators, and indicate that the PI3K/Akt pathway mediates IGF-1 mitogenic signaling during corticogenesis.

Cell-specific localization of the sulphydryl oxidase QSOX in rat peripheral tissues.

Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.

Expression of the sulfhydryl oxidase ALR (Augmenter of Liver Regeneration) in adult rat brain.

Mammalian Augmenter of Liver Regeneration protein (ALR) was first identified as a secondary growth factor involved in liver regeneration. Its sulfhydryl oxidase activity and involvement in iron homeostasis have been recently demonstrated. ALR is expressed in a broad range of peripheral organs, and initial experiments gave also evidence for the occurrence of this protein in brain. In the present study, we investigated in detail the expression of ALR in rat brain sections and determined its cellular and subcellular localizations using biomolecular and immunohistochemical procedures. As shown by Northern blot, ALR is differentially expressed throughout the rat brain, with the highest mRNA levels in the cerebellum and diencephalon. High protein levels were also detected in the brain and cerebellum by Western blot. ALR immunoreactivity was found in neurons and glial cells throughout brain rostrocaudal extent. Labeled astrocytes were particularly abundant in the white matter, and immunoreactive neurons were observed in several regions including the olfactory bulb, isocortex, hippocampal formation, amygdala, thalamus, hypothalamus, some nuclei of the brainstem and cerebellum. In neurons, immunoelectron microscopy showed the protein in the nucleus and mainly in mitochondria. These subcellular localizations may correlate with the occurrence of two ALR protein isoforms in the brain. In the central nervous system, the enzyme might be of importance in heavy metal homeostasis whose dysregulation can induce neurodegenerative disorders.

Ontogenesis of the sulfhydryl oxidase QSOX expression in rat brain.

The spatiotemporal pattern of distribution of the sulfhydryl oxidase QSOX throughout ontogeny was mapped in rat brain using immunohistochemistry. The enzyme was detected on embryonic day (E) 12 in the dawning mantle layer, but the adult-like pattern was acquired postnatally around day 30 (P30). Throughout ontogenesis, rQSOX was detected in immature and mature neurons, but not in glial cells. The rQSOX developmental pattern can be divided into four periods: on E12 the enzyme was detected in the brainstem, more precisely in motoneurons; later (E16), rQSOX-positive cells were also observed in the forebrain, in the caudoputamen, and the subventricular zone. During late embryogenesis (E18-20), the amount of rQSOX cells considerably increased throughout the brain; they initially appeared in the hippocampus, then in the isocortex. From birth onwards, complex modifications of the rQSOX distribution occurred leading to the adult pattern by P30. Although rQSOX exhibits an overall increasing spatiotemporal pattern of distribution, different expression strategies were distinguished depending on the cell type or brain area. By comparing the rQSOX ontogeny with data on neurogenesis and brain histogenesis, we hypothesize that the enzyme could play a role in guiding migrating cells, their settling, and neuronal maturation, e.g., during outgrowth and synaptogenesis.

FAD-linked sulfhydryl oxidase QSOX: topographic, cellular, and subcellular immunolocalization in adult rat central nervous system.

The distribution of the sulfhydryl oxidase QSOX in the rat brain was mapped using immunohistochemistry. QSOX is specifically expressed by neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons express QSOX, different intensities of labeling were observed depending on the area: the strongest labeling was observed in the olfactory bulbs, isocortex, hippocampus, basal telencephalon, several thalamic and hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. This study also describes the ultrastructural localization of QSOX in neuronal cells and demonstrates that the enzyme is associated with the Golgi apparatus. Finally, selected double immunohistochemistry showed that in the hypothalamus the highest levels of QSOX labeling were colocalized in neuron populations that express disulfide-bounded neuropeptides. These observations are consistent with a role of the enzyme in secreted peptide/protein folding. Data presented herein will serve as a basis for further investigations of the physiological function of QSOX in the central nervous system.

Expression of SOx-2, a member of the FAD-dependent sulfhydryl oxidase/quiescin Q6 gene family, in rat brain.

Sulfhydryl oxidases belonging to the FAD-dependent sulfhydryl oxidase/quiescin Q6 family were previously reported in rat peripheral organs but they were not detected in brain. In the present study, by using reverse transcription-polymerase chain reaction and northern blot analysis, we clearly show an ubiquitous expression of the gene in brain; moreover, while only one transcript was present in peripheral organs, at least two transcripts were detected in brain, suggesting a tissue-specific splicing of its mRNA. The shorter one, likely corresponding to the mRNA identified from rat seminal vesicles, was highly expressed in diencephalon and telencephalon. The finding of gene expression in brain is relevant, since its dysregulation could lead to oxidative stress, a causative factor in the pathogenesis of neurodegenerative diseases.