Taro Saito

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

Demonstration of a role for α-synuclein as a functional microtubule-associated protein

α-Synuclein is a major constituent of pathological intracellular inclusion bodies, a common feature of several neu- rodegenerative diseases. Two missense mutations in the α-synuclein gene have been identified in confirmed autosomal-dominant familial Parkinsons disease, which segregate with the illness. However, the physiological function of α-synuclein remains unknown. After biochemical investigations we have revealed tubulin to be an α-synuclein associated/binding protein. Here, we show that α-synuclein induces polymerization of purified tubulin into microtubules. Mutant forms of α-synuclein lose this potential. The binding site of α-synuclein to tubulin is identified, and co-localization of α-synuclein with microtubules is shown in cultured cells. To our knowledge, this is the first demonstration of microtubule-polymerizing activity of α-synuclein. Now we can see a striking resemblance between α-synuclein and tau: both have the same physiological function and pathological features, making abnormal structures in diseased brains known as synucleinopathies and tauopathies. The discovery of a physiological role for α-synuclein may provide a new dimension in researches into the mechanisms of α-synuclein-associated neurodegenerative diseases.

Regulation of Mitochondrial Transport and Inter-Microtubule Spacing by Tau Phosphorylation at the Sites Hyperphosphorylated in Alzheimer's Disease

The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimers disease. Tau is a substrate for protein phosphorylation at multiple sites and occurs in tangles in a hyperphosphorylated state. However, the physiological functions of Tau phosphorylation or how it may contribute mechanistically to Alzheimers pathophysiology are not completely understood. Here, we examined the function of human Tau phosphorylation at three sites, Ser199, Ser202, and Thr205, which together comprise the AT8 sites that mark abnormal phosphorylation in Alzheimers disease. Overexpression of wild-type Tau or mutated forms in which these sites had been changed to either unphosphorylatable alanines or phosphomimetic aspartates inhibited mitochondrial movement in the neurite processes of PC12 cells as well as the axons of mouse brain cortical neurons. However, the greatest effects on mitochondrial translocation were induced by phosphomimetic mutations. These mutations also caused expansion of the space between microtubules in cultured cells when membrane tension was reduced by disrupting actin filaments. Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing. Hyperphosphorylation of the AT8 sites may contribute to axonal degeneration by disrupting mitochondrial transport in Alzheimers disease.

The Regulation of Cyclin-Dependent Kinase 5 Activity through the Metabolism of p35 or p39 Cdk5 Activator

Cyclin-dependent kinase 5 (Cdk5) displays kinase activity predominantly in post-mitotic neurons and its physiological roles are unrelated to cell cycle progression. Cdk5 is activated by its binding to a neuron-specific activator, p35 or p39. The protein amount of p35 or p39 is a primary determinant of the Cdk5 activity in neurons, with the amount of p35 or p39 being determined by its synthesis and degradation. The expression of p35 is induced in differentiated neurons and is enhanced by extracellular stimuli such as neurotrophic factors or extracellular matrix molecules, specifically those acting on the ERK/Erg pathway. p35 is a short-lived protein and its degradation determines the life span. Degradation is mediated by the ubiquitin/proteasome system, similar to that for cyclins in proliferating cells. Autophosphorylation of p35 by Cdk5 is a signal for ubiquitination/degradation, and the degradation of p35 is triggered by glutamate treatment in cultured neurons. p35 is cleaved to p25 by calpain at the time of neuronal cell death, and this limited cleavage is suggested to be the cause of neurodegenerative diseases such as Alzheimer’s disease. Active Cdk5 changes the cellular localization by cleavage of p35 to p25; p35/Cdk5 is associated with membrane or cytoskeletons, but p25/Cdk5 is a soluble protein. Cleavage also increases the life span of p25 and changes the activity or substrate specificity of Cdk5. p25/Cdk5 shows higher phosphorylating activity to tau than p35/Cdk5 in a phosphorylation site-specific manner. Phosphorylation of p35 suppresses cleavage by calpain. Thus, phosphorylation of p35 modulates its proteolytic pattern, stimulates proteasomal degradation and suppresses calpain cleavage. Phosphorylation is age dependent, as p35 is phosphorylated in foetal brains, but unphosphorylated in adult brains. Therefore, foetal phosphorylated p35 is turned over rapidly, whereas adult unphosphorylated p35 has a long life and is easily cleaved to p25 when calpain is activated. p39 is also a short-lived protein and cleaved to the N-terminal truncation form of p29 by calpain. How the metabolism of p39 is regulated, however, is a future problem to be investigated.

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability

Although the roles of cyclin‐dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca2+/calmodulin‐dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome‐dependent degradation of a Cdk5 activator p35, and thus ultimately down‐regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35–/– mice exhibited a lower threshold for induction of long‐term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

Impairment of hippocampal long-term depression and defective spatial learning and memory in p35-/- mice

Cdk5 (cyclin‐dependent kinase 5) activity is dependent upon association with one of two neuron‐specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35–/– mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35–/– mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35–/– mice exhibit impaired long‐term depression and depotentiation of long‐term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5‐dependent phosphorylation state of protein phosphatase inhibitor‐1 was increased in 4‐week‐old mice due to increased levels of p39, which co‐localized with inhibitor‐1 and Cdk5 in the cytoplasm. These results demonstrate that p35‐dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.

Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cycline‐dependent kinase 5 complexes

Cdk5 is a member of the cyclin‐dependent kinases (Cdks), activated by the neuron‐specific activator p39 or p35. The activators also determine the cytoplasmic distribution of active Cdk5, but the mechanism is not yet known. In particular, little is known for p39. p39 and p35 contain localization motifs, such as a second Gly for myristoylation and Lys clusters in the N‐terminal p10 region. Using mutant constructs, we investigated the cellular distribution mechanism. We observed that p39 localizes the active Cdk5 complex in the perinuclear region and at the plasma membrane as does p35. We demonstrated the myristoylation of both p39 and p35, and found that it is a major determinant of their membrane association. Plasma membrane targeting depends on the amino acid sequence containing the Lys‐cluster in the N‐terminal p10 region. In contrast, a non‐myristoylated Ala mutant (p39G2A or p35G2A) showed nuclear localization with stronger accumulation of p39G2A than p35G2A. These results indicate that myristoylation regulates the membrane association of p39 as well as p35 and that the Lys cluster controls their trafficking to the plasma membrane. The differential nuclear accumulation of p39 and p35 suggests their segregated functions, p35–Cdk5 in the cytoplasm and p39–Cdk5 in the nucleus.

Casein kinase 2 is the major enzyme in brain that phosphorylates Ser129 of human α‐synuclein: Implication for α‐synucleinopathies

In Lewy body diseases and multiple system atrophy, α‐synuclein is hyperphosphorylated at Ser129, suggesting a role in pathogenesis. Here, we report purification of the protein kinase in rat brain that phosphorylates Ser129 and its identification as casein kinase‐2 (CK2). We show that most of the activity can be inhibited by heparin, an inhibitor of CK2. Phosphorylated Ser129 was detected in primary cultured neurons and inhibited by CK2 inhibitors. In some cases of Lewy body disease, CK2‐like immunoreactivity was recovered in the sarkosyl‐insoluble fraction, which was enriched in phosphorylated α‐synuclein. Taken together, these findings suggest that CK2 may be involved in the hyperphosphorylation of α‐synuclein in α‐synucleinopathies.

p25/Cyclin‐dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum‐stressed neurons

Dysregulation of cyclin‐dependent kinase 5 (Cdk5) by cleavage of its activator p35 to p25 by calpain is involved in the neuronal cell death observed in neurodegenerative disorders, including Alzheimer’s disease. However, it is not yet clear how p25/Cdk5 induces cell death, although its cytosolic localization or extended half life are thought to be involved. We show here that endoplasmic reticulum (ER) stress causes the calpain‐dependent cleavage of p35 to p25 in primary cultured cortical neurons. Generation of p25 occurred at a cell death execution step in ER‐stressed neurons. p25 translocated to the nucleus in ER‐stressed neurons, whereas p35/Cdk5 was perinuclear in control neurons. Cdk5 inhibitors or dominant‐negative Cdk5 suppressed ER stress‐induced neuronal cell death. These findings indicate that p25/Cdk5 is a proapoptotic factor that promotes ER stress‐induced neuronal cell death in nuclei.

Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser8 and Thr138 as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr138 was dephosphorylated in adult rat, although both Ser8 and Thr138 were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser8 p35 to dual phospho-Ser8/Thr138 p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.