The microtubule-associated protein tau accumulates in Alzheimer’s and other fatal dementias, which manifest when forebrain neurons die. Recent advances in understanding these disorders indicate that brain dysfunction precedes neurodegeneration, but the role of tau is unclear. Here, we show that early tau-related deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic abnormalities caused by the accumulation of hyperphosphorylated tau within intact dendritic spines, where it disrupts synaptic function by impairing glutamate receptor trafficking or synaptic anchoring. Mutagenesis of 14 disease-associated serine and threonine amino acid residues to create pseudohyperphosphorylated tau caused tau mislocalization while creation of phosphorylation-deficient tau blocked the mis-targeting of tau to dendritic spines. Thus, tau phosphorylation plays a critical role in mediating tau mislocalization and subsequent synaptic impairment. These data establish that the locus of early synaptic malfunction caused by tau resides in dendritic spines.

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive-and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4 -2 (neural precursor cell expressed, developmentally downregulated 4 -2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4 -2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4 -2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4 -2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms. The plasma membrane dopamine transporter (DAT) belongs to the family of plasma membrane Na þ /Cl -dependent neurotransmitter transporters, which includes transporters for norepinephrine, serotonin, glycine and GABA (1). The re-uptake of dopamine by DAT is the primary mechanism of termination of dopaminergic signaling in the brain. DAT is also a target of psychostimulants. In the mammalian central nervous system, DAT is expressed exclusively in a small subset of neurons, called dopaminergic neurons, the most prominent of which arise from the substantia nigra and ventral tegmental area and project to the striatum and cerebral cortex. Electron microscopy studies revealed DAT localization in the plasma membrane near the active zone in synapses located along the axonal processes, as well as in various intracellular membrane compartments in the soma and dendrites (2-4).Several lines of evidence, largely generated in heterologous expression cell systems, suggest that DAT expression at the cell surface can be rapidly regulated by endocytosis. For instance, protein kinase C (PKC)-dependent endocytosis of DAT has been observed in various mammalian cell lines (5-9). Phorbol esters also reduce dopamine uptake capacity, DAT transport-associated currents and capacitance measurements in Xenopus oocytes expressing the human DAT (10). Direct evidence for regulated DAT trafficking in neuronal cells is limited to data obtained in vitro using rat brain synaptosomes (11). Down-regulation of surface DAT and the accumulation of DAT in endosomes can be also triggered by amphetamine and other DAT su...

Outbred male Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on their locomotor response to acute cocaine. Concomitant measurement of dopamine clearance in these rats revealed that the differential behavioral responses are associated with the magnitude of dopamine transporter (DAT) inhibition by cocaine. Here, we investigated several factors that might contribute to cocaine-induced behavioral variability and its association with differential inhibition of DAT function. In rats classified as LCRs or HCRs after 10 mg/kg cocaine injection, we found no differences in (1) novelty-induced locomotion, (2) cocaine levels in dorsal striatum or nucleus accumbens (NAc), (3) DAT number or affinity in NAc, or (4) DAT affinity for cocaine in NAc. In rats given 20 mg/kg cocaine, behavior was more uniform across individuals, but still warranted separation into LCR/HCR categories. Additionally, we analyzed the stability of the LCR/HCR classification made during the first test with 10 or 20 mg/kg cocaine by retesting rats 7 days later with saline or cocaine (10 or 20 mg/kg). Before injection, HCRs were more active relative to LCRs and to their own behavior on the first test day. Following cocaine, LCRs and HCRs exhibited similar drug-induced changes in locomotion, but there were unique effects that depended on the cocaine dose given on the first and second test days. Our results argue against several likely explanations for individual differences in cocaine-induced behavior and highlight the influence of a single cocaine exposure on subsequent behavioral responses to the drug.

Background: Recent attention has focused on understanding the role of the brain-reninangiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT 1 ) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice ( Fig. 1).

Termination of dopamine neurotransmission is primarily controlled by the plasma membrane-localized dopamine transporter. In this study, we investigated how this transporter is regulated by tyrosine kinases in neuronal preparations. In rat dorsal striatal synaptosomes, inhibition of tyrosine kinases by genistein or tyrphostin 23 resulted in a rapid (5- amine uptake; the effects were complex with increased maximal velocity but reduced affinity. The facilitatory effect of brain-derived neurotrophic factor on dopamine transporter activity depended on both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Taken together, our results suggest that striatal dopamine transporter function and cell surface expression is constitutively upregulated by tyrosine kinase activation and that brain-derived neurotrophic factor can mediate this type of rapid regulation.

Previously we found that outbred male Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively), based on their open-field locomotor response to acute cocaine (COC; 10 mg/kg, i.p.). Here, we extended this analysis to amphetamine (AMPH; 0.5, 1, and 5 mg/kg, i.p.) and found that the individual differences in behavioral activation were not as pronounced as with COC. This was confirmed with observational analysis of behaviors. Differences in drug-induced activation could involve differential dopamine transporter (DAT) function/trafficking. To address this possibility, we measured [ 3 H]DA uptake into dorsal striatal synaptosomes prepared from rats injected 30 min earlier with saline, COC, or AMPH to determine DAT activity, and radioligand binding to determine the total number of DATs. Striatal [ 3 H]DA uptake in COC-treated HCRs was significantly higher than in LCRs. Furthermore, regardless of LCR/HCR classification, uptake in individual COC-treated rats was significantly correlated with their locomotor behavior in the 30 min after drug administration. In contrast, AMPH-treated rats did not differ in uptake, nor were uptake and locomotor activity correlated. DAT number did not differ between LCRs or HCRs, or between AMPH-treated rats. In addition, when individual differences in COC-induced behavior were no longer detected in LCRs and HCRs 1 week after initial classification, uptake was also similar. Together, these results suggest that a difference in expression of functional DATs on the cell surface contributes to the individual differences observed in COC-induced, but not AMPH-induced, behavioral activation of rats.

Essential for normal movement, the globus pallidus (GP) is a prominent nucleus whose neurons project to all other basal ganglia nuclei. The GP is composed of at least two distinct neuron populations. GP neurons of the rodent contain either the calcium-binding protein parvalbumin (PV) or preproenkephalin (PPE) mRNA, differentially innervate several basal ganglia structures, and have distinct immediate early gene responses to dopamine agonists or antagonists. Recent research has revealed that dopamine directly influences GP neurons, with D2 receptors contributing to both pre- and postsynaptic effects of dopaminergic agents. The existence of D2 mRNA-expressing (D2+) GP neurons has been established, but little is known concerning their numbers, regional distribution, or relationship to pallidal subpopulations identified on the basis of PV immunocytochemistry, PPE mRNA, or axonal targets. Detection of pallidal D2 mRNA with a 35S-cRNA probe revealed that D2+ neurons are found throughout the GP, comprising approximately one-half of pallidal neurons, but they are most dense within a dorsoventral band in lateral GP. While a substantial proportion (42-51%) of all chemically and anatomically labeled pallidal neuron subpopulations expressed D2 transcript, the D2+ neurons exhibited both population-based and regional heterogeneities. Overall, the pallidostriatal cells had a greater density of D2 mRNA than did pallidosubthalamic cells. Also, compared to other pallidal regions, the ventromedial GP contained fewer D2+ cells, and the PPE mRNA-expressing cells in this region had lower densities of D2 mRNA per neuron. These results reveal heterogeneous chemical and anatomical properties of the extensive population of D2+ GP neurons, a potential cellular substrate for dopamine's effects in pallidum.