17 research outputs found

    Elevated Mitochondrial Bioenergetics and Axonal Arborization Size Are Key Contributors to the Vulnerability of Dopamine Neurons

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    SummaryAlthough the mechanisms underlying the loss of neurons in Parkinson’s disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson’s disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP+ (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP+ but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson’s disease is directly due to their particular bioenergetic and morphological characteristics

    Evaluation of D1 and D2 dopamine receptor segregation in the developing striatum using BAC transgenic mice.

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    The striatum is predominantly composed of medium spiny neurons (MSNs) that send their axons along two parallel pathways known as the direct and indirect pathways. MSNs from the direct pathway express high levels of D1 dopamine receptors, while MSNs from the indirect pathway express high levels of D2 dopamine receptors. There has been much debate over the extent of colocalization of these two major dopamine receptors in MSNs of adult animals. In addition, the ontogeny of the segregation process has never been investigated. In this paper, we crossed bacterial artificial chromosome drd1a-tdTomato and drd2-GFP reporter transgenic mice to characterize these models and estimate D1-D2 co-expression in the developing striatum as well as in striatal primary cultures. We show that segregation is already extensive at E18 and that the degree of co-expression further decreases at P0 and P14. Finally, we also demonstrate that cultured MSNs maintain their very high degree of D1-D2 reporter protein segregation, thus validating them as a relevant in vitro model

    Elevated Mitochondrial Bioenergetics and Axonal Arborization Size Are Key Contributors to the Vulnerability of Dopamine Neurons

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    Although the mechanisms underlying the loss of neurons in Parkinson's disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson's disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP(+) (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP(+) but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson's disease is directly due to their particular bioenergetic and morphological characteristics

    Distribution of D1 and D2 reporter proteins in mouse striatal sections reveals extensive segregation.

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    <p>Characterization of tdTomato (D1, red) and GFP (D2, green) immunolabeling in striatal sections prepared from E18, P0 and P14 double-transgenic mice. <b>A</b>: The brain atlas images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067219#pone.0067219-Paxinos1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067219#pone.0067219-Paxinos2" target="_blank">[45]</a> on the left identify the approximate areas where acute slices were obtained. The confocal images shown on the right were obtained using a 60x objective. Although the tdTomato (D1, red) signal was relatively diffuse in E18 and P0 sections, at P14, immunopositive cell bodies could be easily identified. Double-labelled neurons were not frequently detected, as shown in the merge images on the right. <b>B</b>: Summary diagram presenting the results of quantifications performed in striatal sections from P14 animals showing the proportion of total labeled MSNs that express tdTomato (D1), GFP (D2) or both proteins (D1/D2). <b>C</b>–E: Orthogonal distribution of normalized average intensities from single typical D1- (C), D2- (D) and D1/D2-labeled (E) neurons.</p

    Distribution of D1 and D2 reporter proteins in acutely dissociated mouse striatal sections reveals an age-dependent increase in segregation.

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    <p>Characterization of tdTomato (D1, red) and GFP (D2, green) immunolabeling in neurons acutely dissociated from E18, P0 and P14 striatum from double-transgenic mice. <b>A</b>: Examples of acutely dissociated striatal neurons immunolabeled for tdTomato (D1, red) and GFP (D2, green). <b>B–D</b>: Summary diagrams presenting the results of quantifications performed from E18, P0 and P14 mice <b>(</b>tdTomato in B, GFP in C and both proteins in D). <b>E</b>: Single-cell RT-PCR from freshly dissociated P14 WT striatal cells. In this example (wells 2–14), nine collected neurons expressed D1 receptor mRNA and two collected neurons expressed D2L receptor mRNA. One neuron collected expressed both receptors mRNA (well 11). Positive control: whole mesencephalon RNA; negative control: water. <b>F</b>: Table summarizing the results of single-cell RT-PCR experiments performed with P0 and P14 WT mice.</p

    Segregation of D1 and D2 reporter proteins is maintained in postnatal striatal neurons in primary culture.

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    <p>Four types of culture conditions were compared: striatal neurons alone (<b>Mono</b>), striatal neurons cultured with cortical neurons (<b>CoCx</b>), striatal neurons cultured with mesencephalic neurons (<b>CoMs</b>), or mixed cultures containing striatal neurons, mesencephalic neurons and cortical neurons (<b>3x</b>). <b>A</b>: Examples of MSNs in different culture conditions labeled for tdTomato (D1, red) and GFP (D2, green) at 14 days <i>in vitro</i>. <b>B–D</b>: Summary diagrams showing the results of quantifications. <b>B</b>: tdTomato, <b>C</b>: GFP, <b>D</b>: both proteins.</p

    Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells

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    Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells
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