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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 expression in the mouse cerebellum.A) Western blot of TA protein at various postnatal stages (P7, P14, P21, P60). The amount of TA was quantified relative to actin (ACT). The graph shows the quantitative analysis of TA levels at the various ages normalized to P60. TA was highly expressed during postnatal development (P7-P14) and significantly decreased in the adult age. B–B’) Double immunofluorescence of a cerebellar sagittal slice at P14 showing the distribution of TA immunoreactivity (red) and Cb which labels PCs (green). B’ shows a diffuse distribution of TA immunoreactivity in all cerebellar layers (EGL, ML, PCL, IGL and WM) and also in the DCN. C–E) Specificity of the rabbit polyclonal anti-TA antibody by immunostaining. Serial cerebellar sections incubated either with the anti-TA antibody (red, C) or with the anti-TA antibody preincubated with the TA-antigen specific peptide (D–D’). Slices were counterstained with Dapi (white). Some slices were incubated only with the cy3-coniugated anti-rabbit secondary antibody (E). The absence of signal in D’ and E demonstrated the specificity of the anti-TA antibody. All scale bars: 100 µm. One-way ANOVA in A: ** p < 0.01 and *** p < 0.001.
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pone-0068063-g001: TA expression in the mouse cerebellum.A) Western blot of TA protein at various postnatal stages (P7, P14, P21, P60). The amount of TA was quantified relative to actin (ACT). The graph shows the quantitative analysis of TA levels at the various ages normalized to P60. TA was highly expressed during postnatal development (P7-P14) and significantly decreased in the adult age. B–B’) Double immunofluorescence of a cerebellar sagittal slice at P14 showing the distribution of TA immunoreactivity (red) and Cb which labels PCs (green). B’ shows a diffuse distribution of TA immunoreactivity in all cerebellar layers (EGL, ML, PCL, IGL and WM) and also in the DCN. C–E) Specificity of the rabbit polyclonal anti-TA antibody by immunostaining. Serial cerebellar sections incubated either with the anti-TA antibody (red, C) or with the anti-TA antibody preincubated with the TA-antigen specific peptide (D–D’). Slices were counterstained with Dapi (white). Some slices were incubated only with the cy3-coniugated anti-rabbit secondary antibody (E). The absence of signal in D’ and E demonstrated the specificity of the anti-TA antibody. All scale bars: 100 µm. One-way ANOVA in A: ** p < 0.01 and *** p < 0.001.

Mentions: We first performed a time course analysis of TA expression in the mouse cerebellum by western blot. Mice were sacrificed at different ages; P7, P14, P21 and adult (P60). As shown in Figure 1A, TA was highly expressed during the postnatal development at P7 (2.05 ± 0.19; n = 3) and P14 (2.05 ± 0.12; n = 3). At P21 (1.45 ± 0.08, n = 3) the signal decreased to reach half values in the adult age (1 ± 0.12, n = 3) (one-way ANOVA, p < 0.001 and post-hoc Tukey’s test p < 0.001, P7 and P14 vs P21 and P60, p < 0.01 P21 vs P60).


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 expression in the mouse cerebellum.A) Western blot of TA protein at various postnatal stages (P7, P14, P21, P60). The amount of TA was quantified relative to actin (ACT). The graph shows the quantitative analysis of TA levels at the various ages normalized to P60. TA was highly expressed during postnatal development (P7-P14) and significantly decreased in the adult age. B–B’) Double immunofluorescence of a cerebellar sagittal slice at P14 showing the distribution of TA immunoreactivity (red) and Cb which labels PCs (green). B’ shows a diffuse distribution of TA immunoreactivity in all cerebellar layers (EGL, ML, PCL, IGL and WM) and also in the DCN. C–E) Specificity of the rabbit polyclonal anti-TA antibody by immunostaining. Serial cerebellar sections incubated either with the anti-TA antibody (red, C) or with the anti-TA antibody preincubated with the TA-antigen specific peptide (D–D’). Slices were counterstained with Dapi (white). Some slices were incubated only with the cy3-coniugated anti-rabbit secondary antibody (E). The absence of signal in D’ and E demonstrated the specificity of the anti-TA antibody. All scale bars: 100 µm. One-way ANOVA in A: ** p < 0.01 and *** p < 0.001.
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Related In: Results  -  Collection

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pone-0068063-g001: TA expression in the mouse cerebellum.A) Western blot of TA protein at various postnatal stages (P7, P14, P21, P60). The amount of TA was quantified relative to actin (ACT). The graph shows the quantitative analysis of TA levels at the various ages normalized to P60. TA was highly expressed during postnatal development (P7-P14) and significantly decreased in the adult age. B–B’) Double immunofluorescence of a cerebellar sagittal slice at P14 showing the distribution of TA immunoreactivity (red) and Cb which labels PCs (green). B’ shows a diffuse distribution of TA immunoreactivity in all cerebellar layers (EGL, ML, PCL, IGL and WM) and also in the DCN. C–E) Specificity of the rabbit polyclonal anti-TA antibody by immunostaining. Serial cerebellar sections incubated either with the anti-TA antibody (red, C) or with the anti-TA antibody preincubated with the TA-antigen specific peptide (D–D’). Slices were counterstained with Dapi (white). Some slices were incubated only with the cy3-coniugated anti-rabbit secondary antibody (E). The absence of signal in D’ and E demonstrated the specificity of the anti-TA antibody. All scale bars: 100 µm. One-way ANOVA in A: ** p < 0.01 and *** p < 0.001.
Mentions: We first performed a time course analysis of TA expression in the mouse cerebellum by western blot. Mice were sacrificed at different ages; P7, P14, P21 and adult (P60). As shown in Figure 1A, TA was highly expressed during the postnatal development at P7 (2.05 ± 0.19; n = 3) and P14 (2.05 ± 0.12; n = 3). At P21 (1.45 ± 0.08, n = 3) the signal decreased to reach half values in the adult age (1 ± 0.12, n = 3) (one-way ANOVA, p < 0.001 and post-hoc Tukey’s test p < 0.001, P7 and P14 vs P21 and P60, p < 0.01 P21 vs P60).

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