BN-PAGE reveals a stable, non-covalent, ∼110-kDa parkin complex in brain

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A and B) Extracts of brain stem and diencephalon from wild-type and parkin knockout (KO) mice were separated into pellet (P) and supernatant (S) fractions, as described in Materials and methods and . P and S fractions were further fractionated by glycerol gradient centrifugation. Twelve P and 12 S fractions (numbered from the top of the glycerol gradient to the bottom) were analysed by BN-PAGE, followed by Western blotting with either the polyclonal anti-parkin antibody CS2132 (A) or the monoclonal anti-parkin antibody PRK109 (B) The CS2132 antibody detected a 450–550-kDa band in fraction S6, which was also found in parkin-null extract (A). By contrast, the PRK109 antibody (B) revealed a band in fraction S5 at a lower molecular weight (indicated by the arrow), which was absent in parkin knockout brain. The PRK109 antibody also showed some minor, non-specific immunoreactivity at higher molecular weights (indicated by the asterisk) in S5 from both wild-type and parkin knockout. (C) The graph shows how BN gels were calibrated based on the relative mobilities of native protein size markers to determine the molecular weight (MW) of the parkin complex. Markers, denoted by black circles, were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa). The dotted line indicates the estimated MW of the parkin complex. (D) In the left and middle panels, heating brain fraction S5 at 100 °C in 1.5% sodium dodecyl sulphate (SDS) for 10 min immediately prior to BN-PAGE disrupted the ∼110-kDa parkin complex and led to the appearance of monomeric parkin (indicated by the arrow). In the rightmost panel, approximately 50 ng of purified recombinant parkin was analysed by BN-PAGE and Western blot with PRK109 without heat treatment or addition of SDS, revealing a band (indicated by the arrow) with apparent molecular weight (∼50 kDa) consistent with that of monomeric parkin. (E) Omission of Triton X-100 from the extraction protocol did not change the native molecular mass of the parkin complex from brain or the amount of parkin extracted. Numbers to the left of (A), (B), (D) and (E) indicate MWs of the native protein size markers

Comparison of ∼110-kDa complex formation between wild-type (WT) parkin and PD-linked parkin variants

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> COS1 cells were transiently transfected with 70 ng/cm of WT or R256C mutant parkin cDNA, 50 ng/cm of A82E or K161N parkin cDNA, 30 ng/cm of K211N cDNA and 120 ng/cm of R275W cDNA. (A) Transfected COS1 cells were extracted in 1% Triton X-100, followed by glycerol gradient centrifugation of the extracts. Fractions 2–6 of the gradient were analysed by BN-PAGE and parkin immunoblotting to visualize monomer (arrowhead) and ∼110-kDa complex (arrow). (B) In the experiments shown in (A), the amounts of parkin complex and parkin monomer were quantified. The graph represents the amount of parkin complex, expressed as a percentage of the sum of parkin complex and monomer. Asterisks denote significant difference ( < 0.05) from WT. (C and D) In parallel with each of the experiments shown in (A and B), 15 µg of Triton-soluble protein extract was analysed with SDS–PAGE and parkin immunoblotting to compare soluble parkin protein levels between WT and mutant variants. At the low signal intensity of the blots shown in (C), parkin appeared as a doublet due to the presence of an N-terminally truncated parkin species generated through an internal translation initiation site (). No endogenous parkin signal could be observed by SDS–PAGE in untransfected (Untransf.) COS1 cells (C), except after very prolonged film exposures (not shown). The graph in (D) shows SDS–PAGE and Western blot quantification of the Triton-soluble parkin levels. Within each experiment, the level of the parkin mutants was normalized to that of WT parkin. There were no significant differences in soluble parkin levels between WT, A82E, K161N, K211N or R256C ( = 0.96 by one-way )

Requirement for Zebrafish Ataxin-7 in Differentiation of Photoreceptors and Cerebellar Neurons

<div><p>The expansion of a polyglutamine (polyQ) tract in the N-terminal region of ataxin-7 (atxn7) is the causative event in spinocerebellar ataxia type 7 (SCA7), an autosomal dominant neurodegenerative disorder mainly characterized by progressive, selective loss of rod-cone photoreceptors and cerebellar Purkinje and granule cells. The molecular and cellular processes underlying this restricted neuronal vulnerability, which contrasts with the broad expression pattern of atxn7, remains one of the most enigmatic features of SCA7, and more generally of all polyQ disorders. To gain insight into this specific neuronal vulnerability and achieve a better understanding of atxn7 function, we carried out a functional analysis of this protein in the teleost fish <em>Danio rerio</em>. We characterized the zebrafish <em>atxn7</em> gene and its transcription pattern, and by making use of morpholino-oligonucleotide-mediated gene inactivation, we analysed the phenotypes induced following mild or severe zebrafish atxn7 depletion. Severe or nearly complete zebrafish atxn7 loss-of-function markedly impaired embryonic development, leading to both early embryonic lethality and severely deformed embryos. More importantly, in relation to SCA7, moderate depletion of the protein specifically, albeit partially, prevented the differentiation of both retina photoreceptors and cerebellar Purkinje and granule cells. In addition, [1–232] human atxn7 fragment rescued these phenotypes showing strong function conservation of this protein through evolution. The specific requirement for zebrafish atxn7 in the proper differentiation of cerebellar neurons provides, to our knowledge, the first <em>in vivo</em> evidence of a direct functional relationship between atxn7 and the differentiation of Purkinje and granule cells, the most crucial neurons affected in SCA7 and most other polyQ-mediated SCAs. These findings further suggest that altered protein function may play a role in the pathophysiology of the disease, an important step toward the development of future therapeutic strategies.</p> </div

Moderate zebrafish atxn7 depletion impairs the differentiation of cerebellar neurons.

<p>Dorsal views of dissected brains from 5 dpf 1 pmol mmMO<i>zatxn^AUG</i> (A, A’, A’’, A’’’, C, C’ and C’’) and 0.3 pmol MO<i>zatxn7^SPL</i> morphants (B, B’, B’’, B’’’, D, D’ and D’’). DAPI staining (A and B), Pav7 immunostaining of Purkinje cells (A’, B’, C and D) and Vglut1 immunostaining of granule cells (A’’, B’’, C’ and D’). Anterior is to the left. Enlarged views of the brains showed in A’ (C), A’’ (C’), A’’’ (C’’), B’ (D), B’’ (D’), and B’’’ (D’’). Merge images of the photographs A’ and A’’ (A’’’), B’ and B’’ (B’’’), C and C’ (C’’), and D and D’ (D’’).</p

[1–232] human atxn7 fragment can rescue differentiation defects of cerebellar neurons in 0.3 pmol MO<i>zatxn7^SPL</i> morphant.

<p>Dorsal views of dissected brains from 5 dpf 1 pmol mmMO<i>zatxn^AUG</i> (A, A’ and A’’) and 0.3 pmol MO<i>zatxn7^SPL</i> morphants (B, B’ and B’’) and age-matched 0.3 pmol MO<i>zatxn7^SPL</i> morphant co-injected with human <i>atxn7</i> mRNA (2 fmol) (C, C’ and C’’). Pav7 immunostaining of Purkinje cells (A, B and C) and Vglut1 immunostaining of granule cells (A’, B’ and C’). Anterior is to the left. Merge images of the photographs A and A’ (A’’), B and B’ (B’’), and C and C’ (C’’).</p

[1–232] N-terminal fragment of human atxn7 can rescue photoreceptor differentiation defect in 0.3 pmol MO<i>zatxn7^SPL</i> morphant.

<p>Rhodopsin immunostaining (A, B, and C) and DAPI staining (A’, B’, and C’) of eye cryosections of 48 hpf 1 pmol mmMO<i>zatxn7^AUG</i> (A, A’ and A’’) and 0.3 pmol MO<i>zatxn7^SPL</i> morphants (B, B’ and B’’) and age matched 0.3 pmol MO<i>zatxn7^SPL</i> morphant co-injected with 2 fmol human <i>atxn7</i> mRNA (C, C’ and C’’). Merge images of the photographs A and A’ (A’’), B and B’ (B’’), and C and C’ (C’’).</p

Transcription of the zebrafish <i>atxn7</i> gene during development.

<p>(A-F) <i>In situ</i> detection of zebrafish <i>atxn7</i> transcripts on either whole mount embryos at the four-cell stage (A), or 3 (B), 8 (C), 16 (D), and 48 hpf (E) or dissected brain of 120 hpf embryos (F). (I) RT-PCR analysis of zebrafish <i>atxn7</i> transcript accumulation in 1- (1 c), 4- to 16- (4–16 c), and 8- to 64-cell embryos (8–64 c), or 10 (10 h), 24 (24 h), 48 (48 h) and 72 to 96 hpf embryos (3–4 d) and dissected adult brain (Brain), cerebellum (Cer), spinal cord (SC), eye (Eye) and remaining tissues (Ad-brain). RT–PCR for <i>β-actin</i> is shown as a positive control. Abbreviations: Cer, cerebellum; MO, medulla oblongata; TeO, tectum opticum; Tel, telencephalon.</p

Partial zebrafish atxn7 depletion impairs the differentiation of cerebellar neurons.

<p>Dorsal views of dissected brains from 5 (A and D), 6 (B and E), and 7 dpf (C and F) 1 pmol mmMO<i>zatxn7^AUG</i> (A, B, and C) and 0.3 pmol MO<i>zatxn7^SPL</i> morphants (D, E, and F) immunostained with an anti-zebrin II antibody, which specifically labels Purkinje cells.</p

Additional file 2: of Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins

Supplementary Materials and Methods- Alves et al. (DOCX 20 kb

Additional file 1: Fig. S1. of Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins

Lentiviral-mediated overexpression of the Green Fluorescent Protein (GFP) in the mouse cerebellum. Fig. S2. Overexpression of truncated MUT ATXN7 in the mouse cerebellum induces the formation of ubiquitinated ATXN7 aggregates Fig. S3. Lentiviral-mediated overexpression MUT ATXN7 in the mouse cerebellum, at 2 weeks post-infection (early time point). Fig. S4. Phosphorylated TDP-43 expression in the cerebellum of Atxn7 100Q/5Q KI mice. Fig. S5. FUS/TLS is trapped in ATXN7 aggregates in Atxn7 100Q/5Q KI mice. Fig. S6. MBNL1 and MBNL2 immunoreactivity and expression in the cerebellum of Atxn7 100Q/5Q KI mice. (PDF 1053 kb