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The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.

Parfitt DA, Michael GJ, Vermeulen EG, Prodromou NV, Webb TR, Gallo JM, Cheetham ME, Nicoll WS, Blatch GL, Chapple JP - Hum. Mol. Genet. (2009)

Bottom Line: Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional.We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1.Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK.

ABSTRACT
An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

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Sacsin is predominantly expressed in the cell bodies and processes of neurons. Western analysis of mouse and rat tissues was performed using an affinity-purified polyclonal antiserum (r-sacsin4489–4503). (A) r-Sacsin4489–4503 detected a band of >500 kDa in neuronal tissues. The band was not detected when r-sacsin4489–4503 was pre-incubated with immunizing peptide. (B) Sacsin was detected in brain tissue and in testis. (C–Q) In situ hybridization with emulsion autoradiography for SACS mRNA and immunohistochemistry for sacsin protein was performed in rat brain. Sacsin was detected with r-sacsin4489–4503 in D–E, H, J, K, M–O, and with r-sacsin1–17 in P and Q. Layer 5 of the cerebral cortex labelled for SACS mRNA (C) and protein immunoreactivity (D). Larger pyramidal neurons (arrows) had higher expression of both SACS mRNA and sacsin protein than smaller neuronal profiles (arrowhead). (E) Immunohistochemistry showing axonal labelling in the anterior commissure (ac) and heavy neuropil labelling in the nucleus accumbens (Acb). (F) SACS mRNA signal was higher in the anterodorsal thalamus (AD) than the mediodorsal thalamus (MD). The stria medullaris (sm) had no detectable SACS mRNA. (G) Competition control resulted in the loss of specific signal. (H) Immunohistochemistry of the same region showed an identical labelling pattern. (I) Cerebellum in situ hybridization showed high levels of signal over Purkinje cells (Pk), with cells of the granule cell layer (Gc) expressing moderate levels. (J) Immunoreactivity for sacsin was high in Purkinje cells and lower in granule cells. The molecular layer (Mo) had dense immunoreactivity. (L) The magnocellular preoptic nucleus neurons had relatively high levels of autoradiographic signal (arrows) with smaller neurons considerably less labelled (arrowhead). (M) Large neurons (arrow) of the gigantocellular reticular nucleus had high levels of immunoreactivity. Primary dendrites and surrounding fibrous neuropil also labelled. (N) In the medial vestibular nucleus, both large and small neurons and the surrounding fibrous neuropil prominently stained. (O) Large motorneurons in the motor trigeminal nucleus (large arrow) had intense immunoreactivity. Fine, labelled axons (small arrows) in the adjacent motor root of the trigeminal nerve were observed. (P) In layer 5 of the cortex, the N-terminal sacsin antibody stained pyramidal neurons intensely as did the C-terminal antibody (D) but labelled dendritic processes as well. (K and Q) In peptide absorption controls of analogous sections in J and P, respectively, immunoreactivity was significantly reduced. Scale bar = 40 µm in (C and D) and (I–Q); 90 µm in (E–H).
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DDP067F1: Sacsin is predominantly expressed in the cell bodies and processes of neurons. Western analysis of mouse and rat tissues was performed using an affinity-purified polyclonal antiserum (r-sacsin4489–4503). (A) r-Sacsin4489–4503 detected a band of >500 kDa in neuronal tissues. The band was not detected when r-sacsin4489–4503 was pre-incubated with immunizing peptide. (B) Sacsin was detected in brain tissue and in testis. (C–Q) In situ hybridization with emulsion autoradiography for SACS mRNA and immunohistochemistry for sacsin protein was performed in rat brain. Sacsin was detected with r-sacsin4489–4503 in D–E, H, J, K, M–O, and with r-sacsin1–17 in P and Q. Layer 5 of the cerebral cortex labelled for SACS mRNA (C) and protein immunoreactivity (D). Larger pyramidal neurons (arrows) had higher expression of both SACS mRNA and sacsin protein than smaller neuronal profiles (arrowhead). (E) Immunohistochemistry showing axonal labelling in the anterior commissure (ac) and heavy neuropil labelling in the nucleus accumbens (Acb). (F) SACS mRNA signal was higher in the anterodorsal thalamus (AD) than the mediodorsal thalamus (MD). The stria medullaris (sm) had no detectable SACS mRNA. (G) Competition control resulted in the loss of specific signal. (H) Immunohistochemistry of the same region showed an identical labelling pattern. (I) Cerebellum in situ hybridization showed high levels of signal over Purkinje cells (Pk), with cells of the granule cell layer (Gc) expressing moderate levels. (J) Immunoreactivity for sacsin was high in Purkinje cells and lower in granule cells. The molecular layer (Mo) had dense immunoreactivity. (L) The magnocellular preoptic nucleus neurons had relatively high levels of autoradiographic signal (arrows) with smaller neurons considerably less labelled (arrowhead). (M) Large neurons (arrow) of the gigantocellular reticular nucleus had high levels of immunoreactivity. Primary dendrites and surrounding fibrous neuropil also labelled. (N) In the medial vestibular nucleus, both large and small neurons and the surrounding fibrous neuropil prominently stained. (O) Large motorneurons in the motor trigeminal nucleus (large arrow) had intense immunoreactivity. Fine, labelled axons (small arrows) in the adjacent motor root of the trigeminal nerve were observed. (P) In layer 5 of the cortex, the N-terminal sacsin antibody stained pyramidal neurons intensely as did the C-terminal antibody (D) but labelled dendritic processes as well. (K and Q) In peptide absorption controls of analogous sections in J and P, respectively, immunoreactivity was significantly reduced. Scale bar = 40 µm in (C and D) and (I–Q); 90 µm in (E–H).

Mentions: To analyse the expression and localization of sacsin we developed an antiserum (r-sacsin1–17) to the N-terminus of sacsin and an antiserum (r-sacsin4489–4503) to a peptide sequence, which is located near the C-terminus. Western analysis of mouse and rat tissues with r-sacsin4489–4503 detected a specific band of approximately 520 kDa in brain which was competed with immunizing peptide (Fig. 1A and B). r-Sacsin1–17 was unsuitable for western blotting (not shown).


The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.

Parfitt DA, Michael GJ, Vermeulen EG, Prodromou NV, Webb TR, Gallo JM, Cheetham ME, Nicoll WS, Blatch GL, Chapple JP - Hum. Mol. Genet. (2009)

Sacsin is predominantly expressed in the cell bodies and processes of neurons. Western analysis of mouse and rat tissues was performed using an affinity-purified polyclonal antiserum (r-sacsin4489–4503). (A) r-Sacsin4489–4503 detected a band of >500 kDa in neuronal tissues. The band was not detected when r-sacsin4489–4503 was pre-incubated with immunizing peptide. (B) Sacsin was detected in brain tissue and in testis. (C–Q) In situ hybridization with emulsion autoradiography for SACS mRNA and immunohistochemistry for sacsin protein was performed in rat brain. Sacsin was detected with r-sacsin4489–4503 in D–E, H, J, K, M–O, and with r-sacsin1–17 in P and Q. Layer 5 of the cerebral cortex labelled for SACS mRNA (C) and protein immunoreactivity (D). Larger pyramidal neurons (arrows) had higher expression of both SACS mRNA and sacsin protein than smaller neuronal profiles (arrowhead). (E) Immunohistochemistry showing axonal labelling in the anterior commissure (ac) and heavy neuropil labelling in the nucleus accumbens (Acb). (F) SACS mRNA signal was higher in the anterodorsal thalamus (AD) than the mediodorsal thalamus (MD). The stria medullaris (sm) had no detectable SACS mRNA. (G) Competition control resulted in the loss of specific signal. (H) Immunohistochemistry of the same region showed an identical labelling pattern. (I) Cerebellum in situ hybridization showed high levels of signal over Purkinje cells (Pk), with cells of the granule cell layer (Gc) expressing moderate levels. (J) Immunoreactivity for sacsin was high in Purkinje cells and lower in granule cells. The molecular layer (Mo) had dense immunoreactivity. (L) The magnocellular preoptic nucleus neurons had relatively high levels of autoradiographic signal (arrows) with smaller neurons considerably less labelled (arrowhead). (M) Large neurons (arrow) of the gigantocellular reticular nucleus had high levels of immunoreactivity. Primary dendrites and surrounding fibrous neuropil also labelled. (N) In the medial vestibular nucleus, both large and small neurons and the surrounding fibrous neuropil prominently stained. (O) Large motorneurons in the motor trigeminal nucleus (large arrow) had intense immunoreactivity. Fine, labelled axons (small arrows) in the adjacent motor root of the trigeminal nerve were observed. (P) In layer 5 of the cortex, the N-terminal sacsin antibody stained pyramidal neurons intensely as did the C-terminal antibody (D) but labelled dendritic processes as well. (K and Q) In peptide absorption controls of analogous sections in J and P, respectively, immunoreactivity was significantly reduced. Scale bar = 40 µm in (C and D) and (I–Q); 90 µm in (E–H).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667285&req=5

DDP067F1: Sacsin is predominantly expressed in the cell bodies and processes of neurons. Western analysis of mouse and rat tissues was performed using an affinity-purified polyclonal antiserum (r-sacsin4489–4503). (A) r-Sacsin4489–4503 detected a band of >500 kDa in neuronal tissues. The band was not detected when r-sacsin4489–4503 was pre-incubated with immunizing peptide. (B) Sacsin was detected in brain tissue and in testis. (C–Q) In situ hybridization with emulsion autoradiography for SACS mRNA and immunohistochemistry for sacsin protein was performed in rat brain. Sacsin was detected with r-sacsin4489–4503 in D–E, H, J, K, M–O, and with r-sacsin1–17 in P and Q. Layer 5 of the cerebral cortex labelled for SACS mRNA (C) and protein immunoreactivity (D). Larger pyramidal neurons (arrows) had higher expression of both SACS mRNA and sacsin protein than smaller neuronal profiles (arrowhead). (E) Immunohistochemistry showing axonal labelling in the anterior commissure (ac) and heavy neuropil labelling in the nucleus accumbens (Acb). (F) SACS mRNA signal was higher in the anterodorsal thalamus (AD) than the mediodorsal thalamus (MD). The stria medullaris (sm) had no detectable SACS mRNA. (G) Competition control resulted in the loss of specific signal. (H) Immunohistochemistry of the same region showed an identical labelling pattern. (I) Cerebellum in situ hybridization showed high levels of signal over Purkinje cells (Pk), with cells of the granule cell layer (Gc) expressing moderate levels. (J) Immunoreactivity for sacsin was high in Purkinje cells and lower in granule cells. The molecular layer (Mo) had dense immunoreactivity. (L) The magnocellular preoptic nucleus neurons had relatively high levels of autoradiographic signal (arrows) with smaller neurons considerably less labelled (arrowhead). (M) Large neurons (arrow) of the gigantocellular reticular nucleus had high levels of immunoreactivity. Primary dendrites and surrounding fibrous neuropil also labelled. (N) In the medial vestibular nucleus, both large and small neurons and the surrounding fibrous neuropil prominently stained. (O) Large motorneurons in the motor trigeminal nucleus (large arrow) had intense immunoreactivity. Fine, labelled axons (small arrows) in the adjacent motor root of the trigeminal nerve were observed. (P) In layer 5 of the cortex, the N-terminal sacsin antibody stained pyramidal neurons intensely as did the C-terminal antibody (D) but labelled dendritic processes as well. (K and Q) In peptide absorption controls of analogous sections in J and P, respectively, immunoreactivity was significantly reduced. Scale bar = 40 µm in (C and D) and (I–Q); 90 µm in (E–H).
Mentions: To analyse the expression and localization of sacsin we developed an antiserum (r-sacsin1–17) to the N-terminus of sacsin and an antiserum (r-sacsin4489–4503) to a peptide sequence, which is located near the C-terminus. Western analysis of mouse and rat tissues with r-sacsin4489–4503 detected a specific band of approximately 520 kDa in brain which was competed with immunizing peptide (Fig. 1A and B). r-Sacsin1–17 was unsuitable for western blotting (not shown).

Bottom Line: Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional.We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1.Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK.

ABSTRACT
An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

Show MeSH
Related in: MedlinePlus