Image_1_Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.PDF

<p>Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGG^exp) trinucleotides in the 5′UTR of the FMR1 gene encoding fragile X mental retardation protein (FMRP). The patients, with the number of the repeats ranging from 55 to 200, show specific manifestation of clinical symptoms that include intention tremor, gait ataxia, cognitive deficits, and brain atrophy. Accumulation of toxic polyglycine (FMRpolyG), a by-product of the CGG^exp repeat-associated non-ATG (RAN) translation, is considered to be one of the main factors triggering neurodegenerative processes in FXTAS patients. Nevertheless, the nature of the FMRpolyG-induced cell damage, especially in the context of its soluble and inclusion-associated forms, is still elusive. Targeting either biosynthesis, cellular stability or aggregation capacity of toxic FMRpolyG could be considered as a potential therapeutic strategy for FXTAS. Therefore, we tested a variety of quantitative methods based on forced expression of genetic constructs carrying CGG^exp repeats in the context of the FMR1 5′UTR fused to GFP, mCherry or Firefly luciferase gene in or out of frame to the polyglycine encoding sequence. We show that FMRpolyG translation either from native or an AUG-induced start codon as well as the translation yield of the FMRP open reading frame equivalent located downstream of the CGG^exp element can be effectively estimated using fluorescence microscopy, flow cytometry or luciferase assay. We also quantitatively estimated soluble fraction and insoluble form of FMRpolyG aggregated in foci using an electrophoretic separation of cell lysates and fluorescence microscopy, respectively. Importantly, we show that dependent on a fusion tag, FMRpolyG has a different potential for aggregate formation. Our established protocols enable sensitive tracking of FMRP and FMRpolyG quantitative and qualitative changes after treatment with potential therapeutic agents for FXTAS. Furthermore, they can be modified for application to other RAN translation- and aggregation-related diseases.</p

Plectin 1d, 1f, 1b, and 1 link desmin IFs with Z-disks, costameres (DGC), mitochondria, and the outer nuclear/ER membrane system, respectively

<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Representative regions of teased EDL fibers from 4-mo-old f-ple and cKO-ple mice stained for proteins as indicated

Arrowheads and arrows indicate Z-disk–aligned and perpendicular longitudinal desmin-positive costameric structures, respectively. In f-ple fibers, note the colocalization of desmin IFs with syncoilin, synemin, cytokeratin 8, β-DG, dystrophin, nNOS, and syntrophin but not with caveolin 3. In cKO-ple fibers, all costameric marker proteins show profoundly changed localization patterns. Bar, 5 μm. (B and C) Quantitative immunoblotting analysis of gastrocnemius lysates from three 6-mo-old mice per genotype (B) and of microsomal fractions from at least three gel runs (C). Loading was normalized to total protein contents (Coomassie-stained gels). Bar graphs represent mean values ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Longitudinal sections of soleus immunostained using antiserum 46 to plectin

Striated plectin patterns are observed in ple1, ple1b, and dessamples; in ple1d and ple1d/des samples, such patterns are missing. The arrow and arrowheads in the ple1d panel represent plectin-positive sarcolemmal and interior dotlike structures, respectively. Note that the interior of ple1d/des fibers is completely devoid of plectin-positive signals. (B) Teased fibers of EDL were immunostained as in A. Note, the signal associated with longitudinal perinuclear structures was decreased in ple1 compared with ple1b fibers (arrows). Also, costameres were focally disorganized in ple1d and des samples (arrowheads). (C) Ple1d soleus sections double immunolabeled for plectin and desmin (a), desmin and mitochondria (b), or stained for SDH (c). Inset shows subsarcolemmal aggregation of mitochondria in a magnified view of the boxed area. The electron micrograph in panel d shows internal lysis of enlarged mitochondria in the subsarcolemmal region (arrows). (D) Ple1d EDL cross section double immunolabeled for desmin and synemin revealing aggregates in the interior of fibers and largely unaffected sarcolemmal regions (see also inset, a magnified view of the boxed area). (E) Immunofluorescence microscopy of teased ple1d fibers (EDL) using antibodies as indicated. In panels a and b, note the largely unaffected perinuclear and costameric patterns of plectin 1 and 1f, respectively. Panels c and c′ represent sequential confocal sections of one fiber. An optical cross section of this fiber (marked 1) is shown as an inset in panel c′, with horizontal lines indicating the positions of the planes shown in panels c and c′. Note the costameric patterns lacking aggregates in panel c and that desmin aggregates in the interior part of the fiber in panel c′ (arrow). Bars: (A; B; C, a and b; D; and E) 20 μm; (C, c) 50 μm; (C, d) 2 μm. (F) Quantitative immunoblotting of plectin in gastrocnemius lysates from different mouse mutants. Data, relative to WT samples (100%), represent the means ± SEM of three experiments.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Soleus f-ple (a and c) and cKO-ple (b and d) sections double immunolabeled for plectin and desmin (a and b) or stained for desmin alone (c and d)

Note, desmin aggregates in the fiber interior (d, arrow) and accumulates along the sarcolemma (d, arrowhead) in plectin-negative fibers. The double-headed arrow in panel b represents a plectin-positive fiber with a preserved desmin-positive pattern. (B) f-ple (a, c, and e) and cKO-ple (b, d, and f) heart sections immunolabeled using antibodies to proteins as indicated. In cKO-ple cardiomyocytes, note the aggregates of desmin (b, arrow) and misaligned Z-disks (f, inset) as well as the seemingly preserved intercalated disk structures (double arrows). (C) f-ple (a and c) and cKO-ple (b and d) soleus longitudinal (a and b) and EDL cross sections (c and d) stained for proteins as indicated. Asterisks indicate fibers devoid of IFs in the fiber interior. The double-headed arrow in panel b represents a CNF with preserved IF pattern. The dotted boxes in panels c and d indicate areas shown magnified in the insets. (D) Immunofluorescence microscopy of teased fibers from f-ple (a and c) and cKO-ple (b and d) EDL revealing massive longitudinal desmin aggregates (b) and misaligned α-actinin–positive costameres (d, inset) in cKO-ple mice. No misalignments were observed in the case of f-ple costameres (c, inset). Note also the close association of desmin IFs with f-ple nuclei (a, inset) but their detachment from cKO-ple nuclei (b, inset). Dotted boxes indicate areas shown magnified in insets. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p