Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [³H]dopamine uptake kinetics, [³H]CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 μM), but not αPDD, reduced the V_max (∼1 pmol/min/10⁵ cells) for [³H]DA uptake by ∼40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [³H]DA uptake velocity by ∼30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the K_m of [³H]DA uptake (∼200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [³H]CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [³H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function. Synapse 30:79–87, 1998. © 1998 Wiley-Liss, Inc.     

Aberrant phosphorylation of alpha-synuclein in human Niemann-Pick type C1 disease

Niemann-Pick type C1 disease (NPC1) is an autosomal recessive neurovisceral storage disease caused by the mutation of NPC1 gene, resulting in perturbed intracellular transport of unesterified cholesterol. In NPC1, early-onset tauopathy is a constant feature. In addition, in NPC1 patients with ApoE epsilon4 homozygosity, deposition of A beta occurs mimicking Alzheimer disease (AD). Since AD is frequently associated with neuronal expression of alpha-synuclein, we investigated phosphorylated alpha-synuclein (psyn) immunoreactivity in the brains of 12 NPC1 patients, ages at death ranging from 9 months to 55 years. Psyn immunoreactivity was demonstrated in the perikarya of storage neurons and oligodendroglia in 10 cases. The immunoreactivity appeared more intense in subjects who had the ApoE epsilon4 allele. Lewy bodies were found in the substantia nigra in 2 of these cases. The psyn immunoreactivity was most intense in the substantia nigra where tauopathy was most severe. Phosphorylated tau and alpha-synuclein frequently colocalized. This study first documents alpha-synucleinopathy in NPC1. This observation suggests that the defect in intracellular cholesterol trafficking in NPC1 may provoke aberrant phosphorylation of alpha-synuclein and tau, and that this phosphorylation is enhanced by the ApoE epsilon4 allele. Thus, elucidation of metabolic pathways in NPC1 could provide clues to common mechanisms associated with neurodegeneration.     

Tau phosphorylation increases in symptomatic mice overexpressing A30P alpha-synuclein

Mice overexpressing mutant alpha-synuclein develop a progressive loss of motor function associated with the accumulation of aggregated alpha-synuclein in neurons of the brainstem. Recent reports suggest that tau pathology might also be associated with Parkinson disease (PD) and aggregation of alpha-synuclein. We now report that mice overexpressing A30P alpha-synuclein develop abnormally phosphorylated tau in parallel with the accumulation of aggregated alpha-synuclein. Enhanced phosphorylation of tau occurs only in symptomatic mice that also harbor abundant aggregated alpha-synuclein. The increased phosphorylation of tau occurs at S396/404 and S202 as shown by immunoblotting and immunocytochemical studies with the antibodies PHF-1 and AT8. Neurons that accumulated alpha-synuclein occurred in the dorsal brainstem and did not show strong colocalization with neurons that showed abnormal tau phosphorylation, which largely occurred in the ventral brainstem. Aggregation of alpha-synuclein and phosphorylation of tau are associated with increased levels of phosphorylated c-jun kinase (JNK), which is a stress kinase known to phosphorylate tau protein. These results suggest that alpha-synuclein pathology can stimulate early pathological changes in tau.     

Specificity of age differences in emotion regulation

Objectives: The present study attempts to extend previous research examining differences between young and older adults in emotion regulation, by investigating age differences across a great range of facets of emotion regulation.

Method: Young (n = 40) and older adults (n = 40) completed the Difficulties in Emotion Regulation Scale (DERS) and self-report measures of anxiety and depression.

Results: Whereas young and older adults did not differ in terms of acceptance and awareness of emotional responses, younger adults scored higher compared to older overall, indicating greater emotion regulation difficulties. Older adults reported greater ability in engaging in goal-directed behaviour, and refraining from impulsive emotional responses. Increasing age was associated with greater access to emotion regulation strategies and greater clarity of emotions.

Conclusion: Overall, the present cross-sectional results suggest a general stability in late adulthood in several aspects of emotion regulation, suggesting specific adaptations with increasing age. Findings provided support for the construct validity of the DERS and indicated that the relationship between age and emotion regulation is influenced by verbal ability. Current results extend the focus of age-related differences in emotion regulatory control to several theoretically defined forms of emotion regulation.     

Linking alpha-synuclein phosphorylation to reactive oxygen species formation and mitochondrial dysfunction in SH-SY5Y cells

Alpha-synuclein (α-syn) is a soluble protein highly enriched in presynaptic terminals of neurons. Accumulation of α-syn as intracellular filamentous aggregates is a pathological feature of sporadic and familial forms of Parkinson's disease (PD). Changes in α-syn post-translational modifications, as well as mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Here we assessed the correlation between α-syn phosphorylation at serine 129 (Ser129), the formation of reactive oxygen species (ROS) and mitochondrial dysfunction in SH-SY5Y cells expressing A53T mutant or wild-type (WT) α-syn, exposed to ferrous iron (FeSO₄) and rotenone (complex I inhibitor). Under basal conditions, prolonged expression of A53T mutant α-syn altered mitochondria morphology, increased superoxide formation and phosphorylation at Ser129, which was linked to decreased activity of protein phosphatase 2A (PP2A). Exposure to FeSO₄ or rotenone enhanced intracellular ROS levels, including superoxide anions, in both types of cells, along with α-syn Ser129 phosphorylation and mitochondrial depolarization. Most of these changes were largely evident in A53T mutant α-syn expressing cells. Overall, the data suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-syn phosphorylation at Ser129, which may precede cell degeneration in PD.     

In vitro calcium and calmodulin-dependent kinase-mediated phosphorylation of rat brain and spinal cord neurofilament proteins is increased by glycidamide administration

This study was carried out to determine the action of glycidamide (2,3-epoxy-1-propanamide), a neurotoxic metabolite of acrylamide, on Ca²⁺/calmodulin (CaM)-dependent protein kinase phosphorylation of cytoskeletal proteins. Acrylamide has been shown to increase Ca²⁺/CaM-dependent phosphorylation of neurofilament (NF) triplet proteins and autophosphorylation of Ca²⁺/CaM-dependent protein kinase II (CaM kinase II; EC 2.7.1.37). A daily intraperitoneal dose of 0.7 mmol/kg b.wt. of glycidamide or deionized water was administered to male Sprague-Dawley rats. Animals were sacrificed when signs of severe neurotoxicity became apparent at 13–16 days of treatment. Axonal floatation was used to isolate neurofilaments (NFs) and endogenous kinases from brains and spinal cords of treated and control animals. Samples isolated from brain and spinal cord of glycidamide-treated animals showed increased in vitro Ca²⁺/CaM-dependent phosphorylation of endogenous and exogenous NF proteins and increased autophosphorylation of CaM kinase II when compared with controls. CaM binding to the α, β, and β′ subunits of CaM kinase II and antibody binding to the α-subunit of CaM kinase II in brain supernatant isolates was increased as a result of glycidamide treatment. These results suggest that increased Ca^2+/CaM-dependent phosphorylation of cytoskeletal proteins may be involved in the pathogenesis of glycidamide-induced neurotoxicity.     

Serine phosphorylation by casein kinase II controls endocytic L1 trafficking and axon growth

The cell adhesion molecule L1 plays crucial roles in axon tract development. In vitro, L1 presented as a culture substrate stimulates axon elongation by binding to L1 expressed on the growth cone. In migrating growth cones, L1 is endocytosed via the AP-2/clathrin-mediated pathway at the central domain, followed by anterograde vesicular transport and recycling to the plasma membrane of the leading front. It has previously been shown that this endocytic trafficking of L1 is critical for axon elongation (Kamiguchi and Yoshihara [2001] J. Neurosci. 21:9194-9203). Adjacent to the AP-2 recognition site, the L1 cytoplasmic domain has a cluster of acidic amino acids containing Ser1181 that can be phosphorylated by casein kinase II (CKII; Wong et al. [1996a] J. Neurochem. 66:779-786). In this paper, we demonstrate that Ser1181 phosphorylation by CKII is implicated in both normal endocytic trafficking of L1 and L1-stimulated axon growth. Whereas L1 is sorted into transferrin-positive endosomes after endocytosis, pharmacological inhibition of CKII caused some population of L1 to be internalized into transferrin-negative compartments. Single-amino-acid mutations at Ser1181, which either prevent or mimic phosphorylation by CKII, caused similar missorting of internalized L1. Furthermore, dorsal root ganglion neurons that had been treated with a CKII inhibitor or transfected with the L1 mutants showed impaired ability to extend axons on an L1 substrate but not on other control substrates. These results demonstrate the novel role of CKII in L1-mediated axon elongation and stress the importance of functional linkage between L1 phosphorylation and L1 trafficking in migrating growth cones.     

Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D

Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.     

Protein phosphorylation and the regulation of synaptic membrane traffic.

It is well established that protein phosphorylation has an important role in synaptic plasticity. This is achieved, in part, via the presynaptic modulation of neurotransmitter release by protein kinases and protein phosphatases. In recent years, the increase in information available about proteins that are involved in synaptic exocytosis and endocytosis has been exploited in order to study the effects of protein phosphorylation on synaptic-vesicle cycling at the molecular level. The best-characterized protein in this respect is synapsin, whose function in the release of synaptic vesicles from the reserve pool is regulated by phosphorylation. More recently, it has emerged that proteins that function at other stages of the synaptic-vesicle cycle, which include priming of vesicles for docking-fusion and endocytic recycling, are also controlled by phosphorylation. Furthermore, recent work suggests that this regulation of membrane traffic by phosphorylation also occurs postsynaptically, where it contributes to synaptic plasticity.     

Co-association of parkin and alpha-synuclein

Parkin and alpha-synuclein are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Parkin is present in Lewy bodies and axonal spheroids in brains affected by PD, and mutations in parkin cause hereditary forms of Parkinsonism. Alpha-synuclein is a major component of Lewy bodies and is associated with rare cases of PD. We now show that parkin binds to alpha-synuclein, including conditions associated with alpha-synuclein aggregation. Parkin and alpha-synuclein complexes were observed in BE-M17 cells under basal conditions, in BE- M17 cells under oxidative conditions and in brains from control or PD donors. Double staining of PD brains shows parkin and alpha-synuclein co-localize to the same pathological structures (both Lewy bodies and axonal spheroids). These results suggest that parkin interacts with alpha-synuclein and could contribute to the pathophysiology of PD more generally than was previously considered.     

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.     

Specificity of emotion regulation difficulties related to anxiety in early adolescence

Etiological models identify difficulties in emotion regulation as potential contributors to the development and maintenance of anxiety. To date, studies with adolescents have not tested whether different types of anxiety symptoms are related to different emotion regulation difficulties. The current study aimed to examine specificity of associations between emotion regulation difficulties and symptoms of social and generalized anxiety in early adolescence. Ninety adolescents (ages 11–14 years) completed measures of emotion regulation and anxiety symptoms. Social and generalized anxiety symptoms showed similar bivariate correlations with emotion regulation. However, when controlling for generalized anxiety, social anxiety symptoms were uniquely related to emotion understanding, acceptance, evaluation, and reactivity. Generalized anxiety symptoms were uniquely related to emotion modification. The current study suggests that social and generalized anxiety symptoms have both common and unique associations with emotion regulation difficulties in early adolescence, and has implications for which emotion regulation skills to target in clinical interventions.     

Developmental regulation of protein tyrosine phosphorylation in rat brain

Proteins phosphorylated at tyrosine residues in the developing rat brain have been identified with a focus on the nerve growth cone and synaptic terminal. Endogenous protein phosphorylation in membranes from a subcellular growth cone fraction of fetal rat brain revealed prominent 55-60 kD phosphotyrosine-containing proteins. Proteins of similar size were recognized by phosphotyrosine antibodies in isolated growth cone membranes, indicating that they contained phosphotyrosine in vivo. Proteins of 55-60 kD were not highly phosphorylated in synaptosomes from adult brain, suggesting a growth cone-specific function. Generally, tyrosine phosphorylation was much lower in adult brain than in fetal brain fractions. Although some synaptosomal membrane proteins that contained phosphotyrosine corresponded in size with those in growth cone membranes (92 kD, 41 kD), others were unique to synaptosomal membranes (38 kD and 30 kD). Immunoperoxidase staining of fetal rat neocortex with phosphotyrosine antibodies at embryonic day 19 revealed immunoreactivity in presumptive migratory neuroblasts in the intermediate zone and in processes of the molecular layer. Proliferating neuroepithelial cells of the ventricular zone showed little immunoreactivity. Lower levels of phosphotyrosine immunoreactivity were seen until postnatal day 10, correlating with the period of maximal process outgrowth. These results indicate that protein tyrosine phosphorylation in the developing nervous system may be functionally significant in an aspect of neuronal differentiation such as growth cone-mediated process extension and cell migration. An analogous role in the mature brain may be related to synaptic plasticity or function.     

Specificity of relations between adolescents' cognitive emotion regulation strategies and Internalizing and Externalizing psychopathology

OBJECTIVE: Of the study was to examine the extent to which cognitive emotion regulation strategies were 'common determinants' of Internalizing and Externalizing problems and/or 'specific determinants' distinguishing one problem category from the other. METHOD: The sample comprised 271 12- to 18-year-old secondary school students. Internalizing and Externalizing problems were measured by the Youth Self-Report (YSR) and Cognitive Emotion Regulation Strategies were measured by the Cognitive Emotion Regulation Questionnaire (CERQ), in a cross-sectional design. RESULTS: First, adolescents with Internalizing problems, Externalizing problems, comorbid Internalizing and Externalizing problems and a control group were compared on their specific cognitive emotion regulation strategies. Results showed that adolescents with Internalizing problems (both pure and comorbid) scored significantly higher on the cognitive emotion regulation strategies of self-blame and rumination than those with Externalizing (pure) problems or the control group. Unique relationships between the separate cognitive strategies and Internalizing and Externalizing problems were tested by means of Multiple Regression Analyses. Specific relationships were found between Internalizing problems and self-blame, rumination and positive reappraisal and between Externalizing problems and positive refocusing. No 'common' correlates were found. CONCLUSIONS: Theoretical models designed for the prediction of Internalizing problems might not simply be used for the prediction of Externalizing problems. Different (cognitive) intervention strategies should be used for adolescents with Internalizing problems and Externalizing problems.     

Specificity in central myelination: evidence for local regulation of myelin thickness

The ventral funiculi of normal and X-irradiated 13-day-old rats were studied by electron microscopy. In both tissues, oligodendrocytes form myelin sheaths around multiple axons, with a single oligodendrocyte associated with several axons of different sizes. Despite their origin from the same glial cell, the myelin sheaths are thicker for larger axons. Polyribosomes and rough endoplasmic reticulum are observed in distal oligodendrocyte processes, in proximity to the forming myelin sheaths. These results indicate that myelin sheath thickness is matched to axon size via local mechanisms, and suggest a role of polyribosomes and/or rough endoplasmic reticulum in myelin formation.     

Neurofilament phosphorylation: a new look at regulation and function.

Dynamic remodeling of cytoskeleton architecture is necessary for axonal growth and guidance, signal transduction and other fundamental aspects of neuron function. Protein phosphorylation plays a key part in these remodeling processes. Since neurofilaments are major cytoskeletal constituents and are among the most highly phosphorylated neuronal proteins, the control of their behavior serves as a possible model for understanding how phosphorylation regulates the many other phosphoproteins in the cytoskeleton. Recent studies show that neurofilament protein subunits are phosphorylated on both their amino-terminal head domains and carboxy-terminal tails by different protein kinases. This review considers the implications of this complex regulation for neurofilament function in normal neurons and in disease states characterized by neurofibrillary pathology.