The complement of mechanisms underlying tau pathology in neurodegenerative disorders has yet to be elucidated. Among these mechanisms, abnormal tau phosphorylation has received the most attention because neurofibrillary tangles present in Alzheimer’s disease (AD) and related disorders known as tauopathies are composed of hyperphosphorylated forms of this microtubule-associated protein. More recently, we showed that calpain-mediated cleavage leading to the generation of the 17 kDa tau45-230 fragment is a conserved mechanism in these diseases. To obtain insights into the role of this fragment in neurodegeneration, we generated transgenic mice that express tau45-230 and characterized their phenotype. Our results showed a significant increase in cell death in the hippocampal pyramidal cell layer of transgenic tau45-230 mice when compared to wild type controls. In addition, significant synapse loss was detected as early as six months after birth in transgenic hippocampal neurons. These synaptic changes were accompanied by alterations in the expression of the N-methyl-D-aspartate glutamate (NMDA) receptor subunits. Furthermore, functional abnormalities were detected in the transgenic mice using Morris Water Maze and fear conditioning tests. These results suggest that the accumulation of tau45-230 is responsible, at least in part, for neuronal degeneration and some behavioral changes in AD and other tauopathies. Collectively, these data provide the first direct evidence of the toxic effects of a tau fragment biologically produced in the context of these diseases in vertebrate neurons that develop in situ.

We have previously shown that ␤-amyloid (A␤) treatment resulted in an age-dependent calpain activation leading to Tau cleavage into a neurotoxic 17-kDa fragment in a cellular model of Alzheimer disease. This detrimental cellular response was mediated by a developmentally regulated increase in membrane cholesterol levels. In this study, we assessed the molecular mechanisms by which cholesterol modulated A␤-induced Tau cleavage in cultured hippocampal neurons. Our results indicated that these mechanisms did not involve the regulation of the binding of A␤ aggregates to the plasma membrane. On the other hand, experiments using N-methyl-D-aspartic acid receptor inhibitors suggested that these receptors played an essential role in cholesterol-mediated A␤-dependent calpain activity and 17-kDa Tau production. Biochemical and immunocytochemical analyses demonstrated that decreasing membrane cholesterol levels in mature neurons resulted in a significant reduction of the NR1 subunit at the membrane as well as an increase in the number of large NR1, NR2A, and NR2B subunit clusters. Moreover, the majority of these larger N-methyl-D-aspartic acid receptor subunit immunoreactive spots was not juxtaposed to presynaptic sites in cholesterol-reduced neurons. These data suggested that changes at the synaptic level underlie the mechanism by which membrane cholesterol modulates developmental changes in the susceptibility of hippocampal neurons to A␤-induced toxicity. Alzheimer disease (AD)2 is a devastating disorder growing in incidence among the elderly population. The most common form of the disease occurs sporadically; however, some cases are caused by familial genetic mutations (1). Although the cause of each type of AD differs, aging is the greatest factor for both familial and sporadic AD onset (reviewed in Refs. 2, 3). Thus, it is a common goal in the AD field to understand how aging increases neuronal susceptibility to this disease process. Individuals with either form of AD exhibit characteristic morphological changes in the brain, such as senile plaque formation and the assembly of intracellular neurofibrillary tangles (reviewed in Refs. 4 -6). ␤-Amyloid (A␤), the prominent molecular component of senile plaques, has long been suspected as the main initiator of the AD pathogenic cascade (reviewed in Refs. 7-10). One of the means by which A␤ exerts its toxic effects is by inducing post-translational modifications of the Tau protein, such as hyperphosphorylation (reviewed in Refs. 11-14). More recently, we have shown that Tau cleavage was an alternative mechanism by which Tau mediated A␤ toxicity (15). When mature neurons were cultured in the presence of A␤, intracellular calcium (Ca 2ϩ ) concentrations became significantly elevated (16). An increase in intracellular Ca 2ϩ caused the activation of the calpain protease leading to Tau cleavage into the neurotoxic 17-kDa fragment (15, 16). However, A␤ did not initiate this detrimental cascade in young cultured neurons (17). Furthermore, our results indicated that membrane choles...

Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [ 3 H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Shortterm ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function. Dopamine (DA),2 a major central nervous system neurotransmitter, is involved in reward and reinforcing behaviors. DA signaling relies on a critical balance between release and removal of the neurotransmitter within synaptic clefts. Drugs of abuse, including psychostimulants and ethanol, cause maladaptive changes in DA signaling in mesolimbic areas of the brain, leading to addictive behaviors. Localized on pre-synaptic dopaminergic terminals, the dopamine transporter (DAT) is responsible for terminating DA signaling by rapidly removing the transmitter from the synaptic cleft region (1). DAT and other monoamine transporters, including norepinephrine, ␥-amino butyric acid, and serotonin (NET, GAT1, and SERT, respectively) clear extracellular transmitter via a reuptake mechanism (2).Regulation of DAT function is mediated by recycling of the transporters between intracellular compartments and the plasma membrane (3). This dynamic trafficking occurs in both a constitutive and regulated manner to increase or decrease the number of transporters on the cell surface that are available for transmitter reuptake. Trafficking modulators, such as activated protein kinase C (PKC), have been shown to alter basal transporter trafficking rates. PKC-mediated regulation causes an intracellular accumulation of DAT by increasing internalization and decreasing insertion of the transporter on the cell surface (4). In addition to intrinsic transporter modulators, various drugs of abus...

The dysregulation of posttranslational modifications of the microtubule-associated protein (MAP) tau plays a key role in Alzheimer’s disease (AD) and related disorders. Thus, we have previously shown that beta amyloid (Aβ)-induced neurotoxicity was mediated, at least in part, by tau cleavage into the tau45-230 fragment. However, the mechanisms underlying the toxicity of tau45-230 remain unknown. To get insights into such mechanisms, we first determined the subcellular localization of this tau fragment in hippocampal neurons. Tau45-230 was easily detectable in cell bodies and processes extended by these neurons. In addition, cell extraction experiments performed using Triton X-100 and saponin showed that a pool of tau45-230 was associated with the cytoskeleton and the cytoskeleton plus membrane-bound organelles, respectively, in cultured hippocampal neurons. Furthermore, they suggested that these associations were independent of the presence of full-length tau. We also assessed whether this tau fragment could alter axonal transport. Our results indicated that tau45-230 significantly reduced the number of organelles transported along hippocampal axons. This altered axonal transport did not correlate with changes in the total number of organelles present in these cells or in motor protein levels. Together these results suggested that tau45-230 could exert its toxic effects by partially blocking axonal transport along microtubules thus contributing to the early pathology of AD.