The kinase mechanistic target of rapamycin (mTOR) is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and macroautophagic degradation of cellular components. When active, mTOR induces protein translation and inhibits the protein and organelle degradation process of macroautophagy. Accordingly, when blocking mTOR activity with rapamycin, protein translation is blocked and macroautophagy is induced. In the literature, the effects of rapamycin are usually attributed solely to modulation of protein translation, and not macroautophagy. Nevertheless, mTOR also regulates synaptic plasticity directly through macroautophagy, and neurodegeneration may occur when this process is deficient. Macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles, and has been implicated in several human diseases including Alzheimer's, Huntington's and Parkinson's disease. Mice conditionally lacking autophagy-related gene (Atg) 7 function have been exploited to investigate the role of macroautophagy in particular mouse cell populations or entire organs. These studies have revealed that the ability to undergo macroautophagic turnover is required for maintenance of proper neuronal morphology and function. It remained unknown, however, whether it also modulates neurotransmission. We used the Atg7-deficiency model to explore the role of macroautophagy in two sites of the basal ganglia; 1) the dopaminergic neuron, and 2) the direct pathway medium spiny neuron. Briefly, we treated mice with rapamycin, and then examined whether an observed effect was present in control animals, but absent in macroautophagy-deficient lines. We found that rapamycin induces formation of autophagic vacuoles in striatal dopaminergic terminals, and that this is associated with decreased tyrosine hydroxylase (TH)+ axonal profile volumes, synaptic vesicle numbers, and evoked dopamine (DA) release. On the other hand, evoked DA secretion was enhanced and recovery was accelerated in transgenic animals in which the ability to undergo macroautophagy was eliminated in dopaminergic neurons by crossing a mouse line expressing Cre recombinase under the control of the dopamine transporter (DAT) promoter with another in which the Atg7 gene was flanked by loxP sites. Rapamycin failed to decrease evoked DA release or the number of dopaminergic synaptic vesicles per terminal area in the striatum of these mice. Our data demonstrated that mTOR inhibition, specifically through induction of macroautophagy, can rapidly alter presynaptic structure and neurotransmission. We then focused on elucidating the role of macroautophagy in dopaminoceptive neurons, the DA 1 receptor (D1R)-expressing medium spiny neuron. Mice were confirmed to be D1R-specific conditional macroautophagy knockouts as assessed by p62 aggregate accumulation in D1R-rich brain regions (striatum, prefrontal cortex, and the anterior olfactory nuclei), and by analysis of colocalization of Cre recombinase and substance P. Marked age-dependent differences in the presence of p62+ aggregates were noted when comparing the dorsal vs. ventral striatum, and at different ages. We found that the size of striatal postsynaptic densities (PSDs) are modulated by Atg7, as mutant mice have significantly larger PSDs. Surprisingly, we also observed an increase in DAT immunolabel in the dorsal striatum, which suggests that apart from increasing synaptic strength, lack of macroautophagy in postsynaptic neurons could indirectly lead to functional consequences in presynaptic dopaminergic function. Given the newly elucidated role of macroautophagy in modulating a number of pre- and post- synaptic properties, we then explored the potential implications of this process in mediating the effects of synaptic plasticity, specifically to that induced by recreational drugs. An array of studies demonstrates that drugs of abuse induce numerous forms of neuroplasticity in the basal ganglia. Among these changes, rodents that are chronically treated with psychostimulants show increases in dendritic spine density in striatal medium spiny neurons. Little is known about the molecular mechanisms underlying medium spiny neurons gaining more spines in response to psychostimulants. Also, most data, such as involvement of both the D1R and N-methyl-D-aspartic acid (NMDA) receptors, stems from studies using cocaine, and not amphetamine, although a single injection of cocaine has been shown to increase medium spiny neuron spine density, whether acute amphetamine is capable to do so remains to be elucidated. This is an attractive avenue of research to follow given that amphetamines are used recreationally, abused, but unlike cocaine, prescribed for attention deficit hyperactivity disorder and narcolepsy (reviewed in Heal et al., 2013). A myriad of studies has implicated these two proteins in spinogenesis, spine maturation and maintenance, and neuroplasticity. In addition, several studies have demonstrated an association between levels of PSD95 and spine density in various brain regions. Before characterizing the role of mTOR and macroautophagy in psychostimulant-induced plasticity, we examined if an acute injection of amphetamine at multiple doses (1-30 mg/kg) and times of collection after treatment (1-48 hr) influences PSD95 and Homer1b/c in the striatum of wild-type mice by western blotting. We found that amphetamine failed to robustly modify levels of either protein in the striatum. Our data raises several possibilities, including the possibility that unlike cocaine, acute regimens of amphetamine might not regulate spine density in the striatum, and that, it is crucial to examine their effects separately. Finally, this work now provides a starting point to undertake the study of how acute amphetamine affects macroautophagic machinery that regulates molecular, morphological, functional and whole animal behavior.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D85T3HJC |
| Date | January 2014 |
| Creators | Torres, Ciara |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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