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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

Intensity correlation analysis of TA in PC-spine/PF synapses.A–C) Representative ICA analysis of TA and Cb at PC-spine in the z-dimension. A) ICA plots of TA (left) and Cb (right) staining intensities against their respective PDM values (central section). B) Serial sections of PDM images showing positive pixels inside the spine. The image is pseudocolored and a PDM scale bar is shown. C) Positive ICQ values (sign test p < 0.001) at each serial section indicate a dependent distribution of TA and Cb in the PC spine. D–F) Representative ICA analysis of TA and V1 at PF terminals in the z-dimension. D) ICA plots of TA (left) and V1 (right) staining intensities against their respective PDM values (central section). E) Serial sections of PDM images showing positive pixels inside the PF terminal. F) Positive ICQ values (sign test p<0.001) at each serial section indicate a dependent distribution of TA and V1 in the PF. G–I) Representative ICA analysis of Cb-spine and V1-PF terminal in the z-dimension as control. G) ICA plots of Cb (left) and V1 (right) staining intensities against their respective PDM values (central section). H) Serial sections of PDM images showing negative pixels inside the spine. I) negative ICQ values (sign test p < 0.001) at each serial section indicate a segregated distribution of Cb and V1 in the spine. J) Statistical analysis of ICQ values based on multiple contacts. The ICQ values were consistently positive and highly significant not only in the central optical sections but also in the up and down sections for both spines and PF terminals. Accordingly, the ICQ values were consistently and significantly negative along the z-dimension (t-test, p <0.05 relative to 0) in CTR (spine/PF). Of note that the mean values of the three optical sections were not significantly different among them (one-way ANOVA, p < 0.05). Data are represented as mean ± SEM.
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pone-0068063-g007: Intensity correlation analysis of TA in PC-spine/PF synapses.A–C) Representative ICA analysis of TA and Cb at PC-spine in the z-dimension. A) ICA plots of TA (left) and Cb (right) staining intensities against their respective PDM values (central section). B) Serial sections of PDM images showing positive pixels inside the spine. The image is pseudocolored and a PDM scale bar is shown. C) Positive ICQ values (sign test p < 0.001) at each serial section indicate a dependent distribution of TA and Cb in the PC spine. D–F) Representative ICA analysis of TA and V1 at PF terminals in the z-dimension. D) ICA plots of TA (left) and V1 (right) staining intensities against their respective PDM values (central section). E) Serial sections of PDM images showing positive pixels inside the PF terminal. F) Positive ICQ values (sign test p<0.001) at each serial section indicate a dependent distribution of TA and V1 in the PF. G–I) Representative ICA analysis of Cb-spine and V1-PF terminal in the z-dimension as control. G) ICA plots of Cb (left) and V1 (right) staining intensities against their respective PDM values (central section). H) Serial sections of PDM images showing negative pixels inside the spine. I) negative ICQ values (sign test p < 0.001) at each serial section indicate a segregated distribution of Cb and V1 in the spine. J) Statistical analysis of ICQ values based on multiple contacts. The ICQ values were consistently positive and highly significant not only in the central optical sections but also in the up and down sections for both spines and PF terminals. Accordingly, the ICQ values were consistently and significantly negative along the z-dimension (t-test, p <0.05 relative to 0) in CTR (spine/PF). Of note that the mean values of the three optical sections were not significantly different among them (one-way ANOVA, p < 0.05). Data are represented as mean ± SEM.

Mentions: It could be argued that the spatial resolution in the z-dimension of light microscopy was not high enough to distinguish between thin structures. Therefore our colocalization analysis conducted on a single optical section could be affected by signals originating from neighboring structures in the z-dimension. To overcome this problem we performed the intensity correlation analysis [38] between TA staining and the synaptic markers along the z-dimension on single TA positive synaptic contacts. In particular, for each synaptic contact we selected three optical serial sections along the z-dimension (0.3 µm step); then for each section we reported the ICA plots, the PDM images and the ICQ values (see Materials and methods). Briefly, ICA plots identify stained pixel pairs that vary in synchrony, randomly or independently within the cell. Plots for dependent or segregated staining patterns generate hourglass figures that are markedly skewed toward positive or negative values, respectively. In the PDM image each pixel is equal to the PDM value at that location (Figure 7B–E-H). ICQ is a statistically testable quotient which provides an overall index of whether the stained protein pairs are associated in a random (ICQ ~ 0), a dependent (0< ICQ <+0.5) or a segregated manner (0> ICQ > -0.5). The normal approximation of the sign test was used to test if these values were significantly different [38].


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)

Intensity correlation analysis of TA in PC-spine/PF synapses.A–C) Representative ICA analysis of TA and Cb at PC-spine in the z-dimension. A) ICA plots of TA (left) and Cb (right) staining intensities against their respective PDM values (central section). B) Serial sections of PDM images showing positive pixels inside the spine. The image is pseudocolored and a PDM scale bar is shown. C) Positive ICQ values (sign test p < 0.001) at each serial section indicate a dependent distribution of TA and Cb in the PC spine. D–F) Representative ICA analysis of TA and V1 at PF terminals in the z-dimension. D) ICA plots of TA (left) and V1 (right) staining intensities against their respective PDM values (central section). E) Serial sections of PDM images showing positive pixels inside the PF terminal. F) Positive ICQ values (sign test p<0.001) at each serial section indicate a dependent distribution of TA and V1 in the PF. G–I) Representative ICA analysis of Cb-spine and V1-PF terminal in the z-dimension as control. G) ICA plots of Cb (left) and V1 (right) staining intensities against their respective PDM values (central section). H) Serial sections of PDM images showing negative pixels inside the spine. I) negative ICQ values (sign test p < 0.001) at each serial section indicate a segregated distribution of Cb and V1 in the spine. J) Statistical analysis of ICQ values based on multiple contacts. The ICQ values were consistently positive and highly significant not only in the central optical sections but also in the up and down sections for both spines and PF terminals. Accordingly, the ICQ values were consistently and significantly negative along the z-dimension (t-test, p <0.05 relative to 0) in CTR (spine/PF). Of note that the mean values of the three optical sections were not significantly different among them (one-way ANOVA, p < 0.05). Data are represented as mean ± SEM.
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Related In: Results  -  Collection

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pone-0068063-g007: Intensity correlation analysis of TA in PC-spine/PF synapses.A–C) Representative ICA analysis of TA and Cb at PC-spine in the z-dimension. A) ICA plots of TA (left) and Cb (right) staining intensities against their respective PDM values (central section). B) Serial sections of PDM images showing positive pixels inside the spine. The image is pseudocolored and a PDM scale bar is shown. C) Positive ICQ values (sign test p < 0.001) at each serial section indicate a dependent distribution of TA and Cb in the PC spine. D–F) Representative ICA analysis of TA and V1 at PF terminals in the z-dimension. D) ICA plots of TA (left) and V1 (right) staining intensities against their respective PDM values (central section). E) Serial sections of PDM images showing positive pixels inside the PF terminal. F) Positive ICQ values (sign test p<0.001) at each serial section indicate a dependent distribution of TA and V1 in the PF. G–I) Representative ICA analysis of Cb-spine and V1-PF terminal in the z-dimension as control. G) ICA plots of Cb (left) and V1 (right) staining intensities against their respective PDM values (central section). H) Serial sections of PDM images showing negative pixels inside the spine. I) negative ICQ values (sign test p < 0.001) at each serial section indicate a segregated distribution of Cb and V1 in the spine. J) Statistical analysis of ICQ values based on multiple contacts. The ICQ values were consistently positive and highly significant not only in the central optical sections but also in the up and down sections for both spines and PF terminals. Accordingly, the ICQ values were consistently and significantly negative along the z-dimension (t-test, p <0.05 relative to 0) in CTR (spine/PF). Of note that the mean values of the three optical sections were not significantly different among them (one-way ANOVA, p < 0.05). Data are represented as mean ± SEM.
Mentions: It could be argued that the spatial resolution in the z-dimension of light microscopy was not high enough to distinguish between thin structures. Therefore our colocalization analysis conducted on a single optical section could be affected by signals originating from neighboring structures in the z-dimension. To overcome this problem we performed the intensity correlation analysis [38] between TA staining and the synaptic markers along the z-dimension on single TA positive synaptic contacts. In particular, for each synaptic contact we selected three optical serial sections along the z-dimension (0.3 µm step); then for each section we reported the ICA plots, the PDM images and the ICQ values (see Materials and methods). Briefly, ICA plots identify stained pixel pairs that vary in synchrony, randomly or independently within the cell. Plots for dependent or segregated staining patterns generate hourglass figures that are markedly skewed toward positive or negative values, respectively. In the PDM image each pixel is equal to the PDM value at that location (Figure 7B–E-H). ICQ is a statistically testable quotient which provides an overall index of whether the stained protein pairs are associated in a random (ICQ ~ 0), a dependent (0< ICQ <+0.5) or a segregated manner (0> ICQ > -0.5). The normal approximation of the sign test was used to test if these values were significantly different [38].

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