Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity.

Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21.

The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.

Modeling the early stages of Alzheimer's disease by administering intracerebroventricular injections of human native Aß oligomers to rats.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease characterized by the accumulation of aggregated amyloid beta (Aß) and hyperphosphorylated tau along with a slow decline in cognitive functions. Unlike advanced AD, the initial steps of AD pathophysiology have been poorly investigated, partially due to limited availability of animal models focused on the early, plaque-free stages of the disease. The aim of this study was to evaluate the early behavioral, anatomical and molecular alterations in wild-type rats following intracerebroventricular injections of human Aß oligomers (AßOs). Bioactive human AD and nondemented control brain tissue extracts were characterized using ELISA and proteomics approaches. Following a bilateral infusion, rats underwent behavioral testing, including the elevated plus maze, social recognition test, Morris water maze and Y-maze within 6 weeks postinjection. An analysis of brain structure was performed with manganese-enhanced MRI. Collected brain tissues were analyzed using stereology, immunohistochemistry, ELISA and qPCR. No sensorimotor deficits affecting motor performance on different maze tasks were observed, nor was spatial memory disturbed in AD rats. In contrast, a significant impairment of social memory became evident at 21 days postinjection. This deficit was associated with a significantly decreased volume of the lateral entorhinal cortex and a tendency toward a decrease in the total brain volume. Significant increase of cleaved caspase-3-positive cells, microglial activation and proinflammatory responses accompanied by altered expression of synaptic markers were observed in the hippocampus of AD rats with immunohistochemical and qPCR approaches at 6 weeks postinjection. Our data suggest that the social memory impairment observed in AßO-injected rats might be determined by neuroinflammatory responses and synaptopathy. An infusion of native oligomeric Aß in the rat brain represents a feasible tool to model early plaque-free events associated with AD.

In vivo amphetamine action is contingent on αCaMKII.

Addiction to psychostimulants (ie, amphetamines and cocaine) imposes a major socioeconomic burden. Prevention and treatment represent unmet medical needs, which may be addressed, if the mechanisms underlying psychostimulant action are understood. Cocaine acts as a blocker at the transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), but amphetamines are substrates that do not only block the uptake of monoamines but also induce substrate efflux by promoting reverse transport. Reverse transport has been a focus of research for decades but its mechanistic basis still remains enigmatic. Recently, transporter-interacting proteins were found to regulate amphetamine-triggered reverse transport: calmodulin kinase IIα (αCaMKII) is a prominent example, because it binds the carboxyl terminus of DAT, phosphorylates its amino terminus, and supports amphetamine-induced substrate efflux in vitro. Here, we investigated whether, in vivo, the action of amphetamine was contingent on the presence of αCaMKII by recording the behavioral and neurochemical effects of amphetamine. Measurement of dopamine efflux in the dorsal striatum by microdialysis revealed that amphetamine induced less dopamine efflux in mice lacking αCaMKII. Consistent with this observation, the acute locomotor responses to amphetamine were also significantly blunted in αCaMKII-deficient mice. In addition, while the rewarding properties of amphetamine were preserved in αCaMKII-deficient mice, their behavioral sensitization to amphetamine was markedly reduced. Our findings demonstrate that amphetamine requires the presence of αCaMKII to elicit a full-fledged effect on DAT in vivo: αCaMKII does not only support acute amphetamine-induced dopamine efflux but is also important in shaping the chronic response to amphetamine.