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Torsin A Localization in the Mouse Cerebellar Synaptic Circuitry.

Puglisi F, Vanni V, Ponterio G, Tassone A, Sciamanna G, Bonsi P, Pisani A, Mandolesi G - PLoS ONE (2013)

Bottom Line: Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+) ATPase).In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex.These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome Tor Vergata/Laboratory of Neurophysiology and Synaptic Plasticity, Fondazione Santa Lucia, Rome, Italy.

ABSTRACT
Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+) ATPase). To date, a great deal of attention has been focused on neuronal TA since its mutant form causes early-onset (DYT1) torsion dystonia, an inherited movement disorder characterized by sustained muscle contractions and abnormal postures. Interestingly, it has been proposed that TA, by interacting with the cytoskeletal network, may contribute to the control of neurite outgrowth and/or by acting as a chaperone at synapses could affect synaptic vesicle turnover and neurotransmitter release. Accordingly, both its peculiar developmental expression in striatum and cerebellum and evidence from DYT1 knock-in mice suggest that TA may influence dendritic arborization and synaptogenesis in the brain. Therefore, to better understand TA function a detailed description of its localization at synaptic level is required. Here, we characterized by means of rigorous quantitative confocal analysis TA distribution in the mouse cerebellum at postnatal day 14 (P14), when both cerebellar synaptogenesis and TA expression peak. We observed that the protein is broadly distributed both in cerebellar cortex and in the deep cerebellar nuclei (DCN). Of note, Purkinje cells (PC) express high levels of TA also in the spines and axonal terminals. In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex. Finally, TA was observed also in glial cells, a cellular population little explored so far. These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

No MeSH data available.


Related in: MedlinePlus

TA is localized in V2-positive CFs, VGAT-positive BC and PC terminals at P14.Merge of TA (red) and V2 (cyan) staining and of the colocalization mask TA/V2 (mTA/V2 white). TA is localized in the V2-positive CF terminals which impinge on the PC proximal dendrite. B) The negative CTR (CTR-) is represented by the overlapping signal between the V2-positive CFs (cyan) contacting the Cb-positive PC dendrites (green). The panel on the right is the relative colocalization mask (mCb/V2, white). C) Quantitative colocalization analysis of TA in V2-positive terminals. The mean of the TA/V2 overlap coefficients was significantly different from the negative CTR mean value. The insets are high magnification of the white boxes in A and B and show V2 and the relative colocalization masks. D) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT white) in the PCL and ML. TA is localized in the VGAT-positive terminals of inhibitory neurons contacting PC-dendrites and bodies. E) The negative CTR (CTR-) is represented by the overlapping signal between the VGAT-positive terminals (cyan) and the Cb-positive PC bodies (green). VGAT-positive PC collaterals were excluded from the analysis. The panel on the right is the relative colocalization mask (mCb/VGAT, white). F) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in D and E showing VGAT and the relative colocalization masks. G) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT DCN, white) in the DCN region. TA is localized in the VGAT-positive synaptic terminals of PCs which contact DCNs. H) The relative negative control was the overlapping signal of VGAT-positive terminals (cyan) impinging on SMI32-positive DCN bodies (green). The right panel is the relative colocalization mask (m SMI32/VGAT DCN, white). I) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in G and H and show VGAT and the relative colocalization masks. *** p < 0.001. Data are represented as mean± SEM. Scale bars in A-B, D-E and G-H: 10 µm; in C–F–I: 1 µm.
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pone-0068063-g005: TA is localized in V2-positive CFs, VGAT-positive BC and PC terminals at P14.Merge of TA (red) and V2 (cyan) staining and of the colocalization mask TA/V2 (mTA/V2 white). TA is localized in the V2-positive CF terminals which impinge on the PC proximal dendrite. B) The negative CTR (CTR-) is represented by the overlapping signal between the V2-positive CFs (cyan) contacting the Cb-positive PC dendrites (green). The panel on the right is the relative colocalization mask (mCb/V2, white). C) Quantitative colocalization analysis of TA in V2-positive terminals. The mean of the TA/V2 overlap coefficients was significantly different from the negative CTR mean value. The insets are high magnification of the white boxes in A and B and show V2 and the relative colocalization masks. D) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT white) in the PCL and ML. TA is localized in the VGAT-positive terminals of inhibitory neurons contacting PC-dendrites and bodies. E) The negative CTR (CTR-) is represented by the overlapping signal between the VGAT-positive terminals (cyan) and the Cb-positive PC bodies (green). VGAT-positive PC collaterals were excluded from the analysis. The panel on the right is the relative colocalization mask (mCb/VGAT, white). F) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in D and E showing VGAT and the relative colocalization masks. G) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT DCN, white) in the DCN region. TA is localized in the VGAT-positive synaptic terminals of PCs which contact DCNs. H) The relative negative control was the overlapping signal of VGAT-positive terminals (cyan) impinging on SMI32-positive DCN bodies (green). The right panel is the relative colocalization mask (m SMI32/VGAT DCN, white). I) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in G and H and show VGAT and the relative colocalization masks. *** p < 0.001. Data are represented as mean± SEM. Scale bars in A-B, D-E and G-H: 10 µm; in C–F–I: 1 µm.

Mentions: A similar analysis was performed for the PC–CF synapses. The CF is the terminal arbor of the inferior olive neurons. At P14 the multi CFs regression is almost terminated, therefore most of the PCs bear one CF which carries numerous varicosities. In these glutamatergic terminals (cyan, Figure 5A), TA (red, Figure 5A) was abundantly expressed as shown by the colocalization mask TA/V2 (mTA/V2 in Figure 5A,C) and by the quantitative analysis (Figure 5C). In particular, the r index TA/V2 was 0.49 (± 0.014 SEM; n = 197) and was significantly different from the negative CTR (Cb/V2, r = 0.35 ± 0.027 SEM, n = 83; t-test, p < 0.001; Figure 5B,C).


Torsin A Localization in the Mouse Cerebellar Synaptic Circuitry.

Puglisi F, Vanni V, Ponterio G, Tassone A, Sciamanna G, Bonsi P, Pisani A, Mandolesi G - PLoS ONE (2013)

TA is localized in V2-positive CFs, VGAT-positive BC and PC terminals at P14.Merge of TA (red) and V2 (cyan) staining and of the colocalization mask TA/V2 (mTA/V2 white). TA is localized in the V2-positive CF terminals which impinge on the PC proximal dendrite. B) The negative CTR (CTR-) is represented by the overlapping signal between the V2-positive CFs (cyan) contacting the Cb-positive PC dendrites (green). The panel on the right is the relative colocalization mask (mCb/V2, white). C) Quantitative colocalization analysis of TA in V2-positive terminals. The mean of the TA/V2 overlap coefficients was significantly different from the negative CTR mean value. The insets are high magnification of the white boxes in A and B and show V2 and the relative colocalization masks. D) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT white) in the PCL and ML. TA is localized in the VGAT-positive terminals of inhibitory neurons contacting PC-dendrites and bodies. E) The negative CTR (CTR-) is represented by the overlapping signal between the VGAT-positive terminals (cyan) and the Cb-positive PC bodies (green). VGAT-positive PC collaterals were excluded from the analysis. The panel on the right is the relative colocalization mask (mCb/VGAT, white). F) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in D and E showing VGAT and the relative colocalization masks. G) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT DCN, white) in the DCN region. TA is localized in the VGAT-positive synaptic terminals of PCs which contact DCNs. H) The relative negative control was the overlapping signal of VGAT-positive terminals (cyan) impinging on SMI32-positive DCN bodies (green). The right panel is the relative colocalization mask (m SMI32/VGAT DCN, white). I) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in G and H and show VGAT and the relative colocalization masks. *** p < 0.001. Data are represented as mean± SEM. Scale bars in A-B, D-E and G-H: 10 µm; in C–F–I: 1 µm.
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pone-0068063-g005: TA is localized in V2-positive CFs, VGAT-positive BC and PC terminals at P14.Merge of TA (red) and V2 (cyan) staining and of the colocalization mask TA/V2 (mTA/V2 white). TA is localized in the V2-positive CF terminals which impinge on the PC proximal dendrite. B) The negative CTR (CTR-) is represented by the overlapping signal between the V2-positive CFs (cyan) contacting the Cb-positive PC dendrites (green). The panel on the right is the relative colocalization mask (mCb/V2, white). C) Quantitative colocalization analysis of TA in V2-positive terminals. The mean of the TA/V2 overlap coefficients was significantly different from the negative CTR mean value. The insets are high magnification of the white boxes in A and B and show V2 and the relative colocalization masks. D) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT white) in the PCL and ML. TA is localized in the VGAT-positive terminals of inhibitory neurons contacting PC-dendrites and bodies. E) The negative CTR (CTR-) is represented by the overlapping signal between the VGAT-positive terminals (cyan) and the Cb-positive PC bodies (green). VGAT-positive PC collaterals were excluded from the analysis. The panel on the right is the relative colocalization mask (mCb/VGAT, white). F) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in D and E showing VGAT and the relative colocalization masks. G) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT DCN, white) in the DCN region. TA is localized in the VGAT-positive synaptic terminals of PCs which contact DCNs. H) The relative negative control was the overlapping signal of VGAT-positive terminals (cyan) impinging on SMI32-positive DCN bodies (green). The right panel is the relative colocalization mask (m SMI32/VGAT DCN, white). I) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in G and H and show VGAT and the relative colocalization masks. *** p < 0.001. Data are represented as mean± SEM. Scale bars in A-B, D-E and G-H: 10 µm; in C–F–I: 1 µm.
Mentions: A similar analysis was performed for the PC–CF synapses. The CF is the terminal arbor of the inferior olive neurons. At P14 the multi CFs regression is almost terminated, therefore most of the PCs bear one CF which carries numerous varicosities. In these glutamatergic terminals (cyan, Figure 5A), TA (red, Figure 5A) was abundantly expressed as shown by the colocalization mask TA/V2 (mTA/V2 in Figure 5A,C) and by the quantitative analysis (Figure 5C). In particular, the r index TA/V2 was 0.49 (± 0.014 SEM; n = 197) and was significantly different from the negative CTR (Cb/V2, r = 0.35 ± 0.027 SEM, n = 83; t-test, p < 0.001; Figure 5B,C).

Bottom Line: Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+) ATPase).In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex.These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome Tor Vergata/Laboratory of Neurophysiology and Synaptic Plasticity, Fondazione Santa Lucia, Rome, Italy.

ABSTRACT
Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+) ATPase). To date, a great deal of attention has been focused on neuronal TA since its mutant form causes early-onset (DYT1) torsion dystonia, an inherited movement disorder characterized by sustained muscle contractions and abnormal postures. Interestingly, it has been proposed that TA, by interacting with the cytoskeletal network, may contribute to the control of neurite outgrowth and/or by acting as a chaperone at synapses could affect synaptic vesicle turnover and neurotransmitter release. Accordingly, both its peculiar developmental expression in striatum and cerebellum and evidence from DYT1 knock-in mice suggest that TA may influence dendritic arborization and synaptogenesis in the brain. Therefore, to better understand TA function a detailed description of its localization at synaptic level is required. Here, we characterized by means of rigorous quantitative confocal analysis TA distribution in the mouse cerebellum at postnatal day 14 (P14), when both cerebellar synaptogenesis and TA expression peak. We observed that the protein is broadly distributed both in cerebellar cortex and in the deep cerebellar nuclei (DCN). Of note, Purkinje cells (PC) express high levels of TA also in the spines and axonal terminals. In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex. Finally, TA was observed also in glial cells, a cellular population little explored so far. These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

No MeSH data available.


Related in: MedlinePlus