Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners

Cyclin‐dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5‐null Purkinje cells and Purkinje cells in Wnt1^cre‐mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7^cre‐mediated p35; p39 double KO (L7^cre‐p35^f/f; p39^–/–) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell‐autonomous in vivo . We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5‐null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non‐cell‐autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain‐derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7^cre‐p35^f/f; p39^–/– mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175–1187, 2017     

Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.     

Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum

Cyclin‐dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron‐specific activating subunit. It was previously reported that Cdk5^−/− mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5^−/− embryos. To further study this abnormal development, we generated and analyzed Dlx5/6‐Cre p35 conditional KO (cKO); p39^−/− mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6‐Cre p35cKO; p39^−/− mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6‐Cre p35cKO; p39^−/− mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell‐autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule‐associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migra tion. Previous in vitro phosphorylation experiments revealed that cyclin‐dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5?/? mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP‐DCX‐WT, or GFP‐DCX‐S332A and analyzed neurite extension and migration. Introduction of GFP‐DCX‐WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP‐DCX‐S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5?/? brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 929–942, 2010     

CDK5 phosphorylates eNOS at Ser‐113 and regulates NO production

Phosphorylation of endothelial nitric oxide synthase (eNOS) is key mechanism in response to various forms of cellular stimulation. Through protein nitration by peroxynitrite, eNOS is believed to be responsible for the major abnormalities in several important neurodegenerative diseases including Alzheimer's (AD) and Parkinson's diseases (PD). Recent studies provide important in vivo evidence that hyperactivation of Cdk5 by p25 plays an essential role in the cell death of neurons in experimental models of AD and PD. This study focuses on the functional regulation of eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. Our results showed that Cdk5 can phosphorylate eNOS both in vitro and in vivo. In vitro kinase assay together with the bioinformatic analysis and site direct mutagenesis revealed that Ser‐113 is the major phosphorylation site for Cdk5. Most interestingly, the nitrite production was significantly reduced in eNOS and Cdk5/p35 co‐transfected SH‐SY5Y cells when compared with co‐transfection of Cdk5/p35 and S113A. Together, our data suggest that Cdk5 can phosphorylate eNOS at the Ser‐113 site and down‐regulate eNOS‐derived NO levels. J. Cell. Biochem. 110: 112–117, 2010. © 2010 Wiley‐Liss, Inc.     

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.     

Protein-protein interactions in Cdk5 regulation and function

Cdk5 is a unique member of the cyclin-dependent kinase (Cdk) family of small protein kinases. In association with its neuron-specific activator p35 or p39, Cdk5 displays many regulatory properties distinct from other Cdks. A growing body of evidence has suggested that Cdk5-p35 has important implications in a variety of neuronal activities occurring in the central nervous system. In brain, Cdk5-p35 appears to exist as large molecular complexes with other proteins, and protein-protein interactions appear to be a molecular principle for Cdk5-p35 to conduct its physiological functions. Over the past decade, a number of proteins have been identified to associate with Cdk5-p35. While the majority of these proteins mediate their interaction with Cdk5 through p35, implying that p35 may act not only as an activator of Cdk5 but also as an adaptor to associate Cdk5 with its regulators and physiological targets, a small group of other proteins are found to link directly with Cdk5. In addition, Cdk5 has been found to phosphorylate a diverse list of substrates, further implicating its regulatory roles in a wide range of cellular processes. In this review, we present an updated inventory of the interacting proteins of Cdk5-p35 kinase and its substrates as well as a discussion on the implicated effects of these interactions.     

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia,Is Essential for Oligodendroglia Differentiation and Myelin Repair

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39^−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair.     

Cdk9/Cyclin T1 complex: a key player during the activation/differentiation process of normal lymphoid B cells

Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types. Limited data are available regarding the expression of Cdk9/Cyclin T1 in hematopoietic and lymphoid tissues. Cdk9/Cyclin T1 expression seems to be related to particular stages of lymphoid differentiation/activation. In this study, we observed that the expression level of Cdk9/Cyclin T1 in vivo increases in memory B cells compared to naïve B cells, and in activated B cells, compared to non‐activated ones. The expression level of the Cdk9/Cyclin T1 complex does not increase in cells induced to differentiate in vitro. In addition, we showed that Cdk9 interacts with E12 and E47, specifically activated during Germinal Center (GC) reaction. Taken together this data suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation through germinal center reaction. J. Cell. Physiol. 215: 276–282, 2008. © 2008 Wiley‐Liss, Inc.     

Early Postnatal In Vivo Gliogenesis From Nestin-Lineage Progenitors Requires Cdk5

The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5’s function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5^flox/flox [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5^wt/wt [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.     

Coordination of proliferation and neuronal differentiation by the retinoblastoma protein family

Once neurons enter the post‐mitotic G0 phase during central nervous system (CNS) development, they lose their proliferative potential. When neurons re‐enter the cell cycle during pathological situations such as neurodegeneration, they undergo cell death after S phase progression. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are highly coordinated. In this review, the coordination of cell cycle control and neuronal differentiation during development are discussed, focusing on regulation by the Rb family of tumor suppressors (including p107 and p130), and the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors. Based on recent findings suggesting roles for these families in regulating neurogenesis and neuronal differentiation, I propose that the Rb family is essential for daughter cells of neuronal progenitors to enter the post‐mitotic G0 phase without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family regulates the timing of neuronal progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina. Rb's lack of involvement in regulating the initiation of neuronal differentiation may explain why Rb family‐deficient retinoblastomas characteristically exhibit neuronal features.     

Loss of mitofusin 2 links beta‐amyloid‐mediated mitochondrial fragmentation and Cdk5‐induced oxidative stress in neuron cells

Mitochondrial dysfunction is implicated in age‐related degenerative disorders such as Alzheimer's disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Aβ oligomers (AβOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AβOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin‐dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphology‐related proteins in Neuro‐2a (N2a) cells stably expressing the Swedish mutation of amyloid precursor protein (APP), which is known to increase AβO production. We demonstrated that mitochondrial fragmentation by AβOs accompanies reduced mitofusin 1 and 2 (Mfn1/2) levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2‐related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, over‐expression significantly inhibits the AβO‐mediated cell death pathway. Therefore, these results indicate that AβO‐mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5‐induced Prx2 phosphorylation.

     

The Expression of Cdk5, p35, p39, and Cdk5 Kinase Activity in Developing, Adult, and Aged Rat Brains

The expression of cyclin-dependent kinase 5 (Cdk5) and its regulatory subunits, p35 and p39, was investigated in rat brain from embryonic day 12 (E12) to postnatal 18 months (18M). The Cdk5 protein levels increased from E12 to postnatal day 7 (P7) and remained at this level until 18M. The Cdk5 kinase activity and the levels of both p35 mRNA and protein were low at E12, became prominent at E18-P14 but then decreased in the adult and aged rat brains of 3M to 18M. In comparison, the expression pattern of p39 appeared to have an inverse relationship to that of Cdk5 and p35. In regional distribution studies, p35 protein levels and Cdk5 kinase activity were significantly higher in the cerebral cortex and hippocampus, but lower in the cerebellum and striatum. These results suggested that Cdk5, p35 and p39 might have region-specific and developmental stage-specific functions in rat brain.