Splice Isoforms of the Polyglutamine Disease Protein Ataxin-3 Exhibit Similar Enzymatic yet Different Aggregation Properties

Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively). In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5′ variants and both of the known 3′ ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity

Health, education, and social care provision after diagnosis of childhood visual disability

Aim: To investigate the health, education, and social care provision for children newly diagnosed with visual disability.Method: This was a national prospective study, the British Childhood Visual Impairment and Blindness Study 2 (BCVIS2), ascertaining new diagnoses of visual impairment or severe visual impairment and blindness (SVIBL), or equivalent vi-sion. Data collection was performed by managing clinicians up to 1-year follow-up, and included health and developmental needs, and health, education, and social care provision.Results: BCVIS2 identified 784 children newly diagnosed with visual impairment/SVIBL (313 with visual impairment, 471 with SVIBL). Most children had associated systemic disorders (559 [71%], 167 [54%] with visual impairment, and 392 [84%] with SVIBL). Care from multidisciplinary teams was provided for 549 children (70%). Two-thirds (515) had not received an Education, Health, and Care Plan (EHCP). Fewer children with visual impairment had seen a specialist teacher (SVIBL 35%, visual impairment 28%, χ2p < 0.001), or had an EHCP (11% vs 7%, χ2p < 0 . 01).Interpretation: Families need additional support from managing clinicians to access recommended complex interventions such as the use of multidisciplinary teams and educational support. This need is pressing, as the population of children with visual impairment/SVIBL is expected to grow in size and complexity.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Toward understanding the role of protein context in the polyglutamine disease, SCA3

The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG) n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG) n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis

Ataxin-3 is alternatively spliced in <i>ATXN3</i> YAC transgenic and human brain.

<p>(A) Schematic representation of the <i>ATXN3</i> gene showing exons that encode specific functional domains. Untranslated regions (U) are not drawn to scale. The splicing pattern of the originally identified 2UIM ataxin-3 transcript is shown below, while above is shown the alternative splicing that links exon 10 to exon 11, generating 3UIM ataxin-3. Asterisks indicate exons that encode amino acids comprising the catalytic triad, polyQ denotes the polyglutamine domain, and the arrowhead indicates a polymorphic Tyr/Stop-encoding residue within the hydrophobic domain (Φ) of the C-terminus of 2UIM ataxin-3. C-terminal amino acid sequences are shown below the diagram, beginning with shared sequence in both isoforms extending from the polyQ domain, followed by the divergent sequences for the 2UIM and 3UIM isoforms; residues omitted in some SNP variants of the 2UIM isoform are shown in grey. (B) Diagram showing 5′ ataxin-3 splice variants identified and confirmed by sequencing. Multiple variants are detectable in mature mRNA from adult murine brain (and fetal brain, data not shown) by RT-PCR, using primers targeting the 5′UTR/exon 1 junction and exon 9 (arrows). All identified splice variants that maintain the open reading frame eliminate at least one catalytic triad residue, and thus are not likely to encode an active DUB. Darkly shaded areas are downstream of a frameshift-induced stop codon. (C–D) Endogenous <i>Atxn3</i> and transgenic <i>ATXN3</i> “full length” splice variants were amplified by RT-PCR using species-specific (human, hum; murine, ms) and sequence-specific (10-exon 2UIM-encoding, 10; 11-exon 3UIM-encoding, 11) primers. 10-exon and 11-exon variants are both detectable in mature mRNA: (C) endogenous <i>Atxn3</i> from all murine samples and unexpanded <i>ATXN3</i> from MJD15.4(+/−) brain; and (D) expanded <i>ATXN3</i> from MJD84.2(+/−) brain, and unexpanded <i>ATXN3</i> from pooled human brain tissue (hum). Perinatal day 1–3 (P_Q84), adult (A), or fetal (F) sources were used, as indicated. Note: Primers are not drawn to scale; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013695#s2" target="_blank">Materials and Methods</a> for exact sequences and locations.</p

2UIM and 3UIM ataxin-3 display similar DUB activity against defined ubiquitin chains <i>in vitro</i>.

<p>(A–C) Recombinant GST-ATXN3(Q22) (3UIM or 2UIM) can cleave K48-linked hexaubiquitin (A), K63-linked tetraubiquitin (B), and mixed linkage K48-K63-K48 tetraubiquitin (C) chains. Results with catalytically inactive GST-ataxin-3 (C14A mutant) are also shown. (D–E) Recombinant 2UIM and 3UIM GST-ATXN3(Q22) cleave Ub-AMC at a similar rate. (D) Ub-AMC reaction curves. Both 3UIM and 2UIM ataxin-3 area able to cleave Ub-AMC, while reactions with either an unrelated control protein (the non-DUB F-box protein FBXO2) or buffer only show no cleavage. Error bars show standard deviations. (E) There is no significant difference between the initial reaction velocity of 2UIM and 3UIM ataxin-3(Q22) (p>0.4 by a 2 tailed heteroscedastic Student's t-test).</p

2UIM ataxin-3 is more prone to aggregate than 3UIM ataxin-3.

<p>(A) Representative immunofluorescence of Cos7 cells transiently expressing Flag-tagged ataxin-3(Q22) splice isoforms or the UIM3(SA/DG) mutant. Cells were gated by fluorescence intensity into populations of moderate and high expressors. (B) Quantification of puncta per cell in (A). Error bars represent the standard deviation within each bin. Frequency distributions differ significantly between ATXN3(Q22)2UIM and ATXN3(Q22)3UIM and between ATXN3(Q22)2UIM and ATXN3(Q22)UIM3(SA/DG) mutant ataxin-3 (*p<0.0001, ** p<1×10^-11), but not between ATXN3(Q22)3UIM and ATXN3(Q22)UIM3(SA/DG) mutant ataxin-3 by a χ² test for independence, df  = 3. (C) Supernatant (sup) and pellet (pel) fractions of non-denaturing RIPA brain lysates from aged MJD84.2 (ATXN3(Q84)3UIM) and Q71B (ATXN3(Q71)2UIM) hemizygous transgenic mice were analyzed by Western blot with 1H9 anti-ataxin-3 antibody. Insoluble microaggregates are detected at the base of lane wells, whereas soluble transprotein and endogenous ataxin-3 are visualized within the resolving gel. (D) Quantification of the ratio of insoluble to soluble ataxin-3 transprotein seen in (C). 3UIM-predominant MJD84.2 mice show a significantly lower ratio of insoluble:soluble transprotein than 2UIM-only Q71B mice (*p<0.0005 by a 1 tailed heteroscedastic Student's t-test).</p

Model for the differential aggregation properties and processing of 2UIM and 3UIM ataxin-3.

<p>In the absence of polyglutamine expansion, 3UIM ataxin-3 follows a multi-domain aggregation mechanism to generate limited oligomeric species without detectable formation of SDS-insoluble fibrillar aggregates. In contrast, 2UIM ataxin-3 exists in at least two monomeric states: the native conformation, in which the hydrophobic tail remains buried and protected from the aqueous environment, and an aggregation-prone conformation with an exposed hydrophobic tail. The aggregation prone monomer can revert to the native conformation or oligomerize through both the self-association propensity of the Josephin domain (like 3UIM ataxin-3) and hydrophobic interactions of the 2UIM-specific domain. Within 2UIM oligomers, the hydrophobic C-termini will associate, increasing the local polyglutamine concentration beyond that seen in 3UIM oligomers, favoring formation of detergent-insoluble aggregates. Unstable forms of monomer and oligomer are subject to protein quality control mechanisms, including proteasomal degradation for 2UIM ataxin-3. Insoluble fibrils, which are less well handled by protein quality control systems, accumulate as biochemically and microscopically detectable aggregates.</p