To study the plausible pathogenesis of SCA8, we cloned the ATXN8OS cDNA containing spliced exons D, C2, C1, B, and A [57].
Sequence analysis revealed the existence of a 102 amino-acid ORF1 and a 41 amino acids plus a polyleucine tract ORF2 in ATXN8OS RNA (Fig.
8A). To investigate if these two ORFs can be translated via a
cap-independent IRES activity, we constructed a dicistronic vector pRF in which firefly luciferase was placed after the Renilla luciferase (Fig.
8B). The expression construct was under the control of the HSV-TK promoter. Regions upstream of ATXN8OS ORF1 and ORF2 (Fig. 9A) were inserted into the intercistronic region of the pRF. The IRES from the encephalomyocarditis virus (ECMV) was inserted as a positive control.
By comparing the expression levels of firefly luciferase and Renilla luciferase among these constructs after transfection, we could possibly define the IRES activity of ATXN8OS. Transient transfection in HEK-293 cells and reporter assay revealed the presence of bipartite
cap-independent IRESs in the ATXN8OS RNA. When the expressed luciferase level of the ECMV IRES was set as 100%, the ATXN8OS 5’
fragments 1~1032, 1~395, and 396~1032 directed firefly luciferase synthesis to a level of 23.5 %, 33.7 %, and 22.5 %, respectively, as
compared to the ECMV IRES sequences (Fig. 9B). Both non-overlapping 1~395 and 396~1032 fragments displayed IRES activity; the presence of multiple internal ribosome entry sites was indicated. As the ATXN8OS 5’
fragment 1~395 expressed significant higher level of relative luciferase
38
activity as compared to fragment 1~1032 (p = 0.024) and fragment 396~1032 (p = 0.014). The results suggest the possible IRES activity existing in the 5’ regions upstream of ATXN8OS ORF1 and ORF2.
39
Discussion
SCA8 was first proposed to be caused by an RNA gain-of-function mechanism and analysis of ATXN8OS sequence did not reveal any possible spliced isoform possessing an ORF to extend through the expansion [64]. In this study, we revealed the presence of bipartite cap-independent IRESs in the ATXN8OS RNA. The lower luciferase activity of fragment 1~1032 was observed when compared with fragment 1~395 in ATXN8OS RNA. This result may be due to the specialized structure of different RNA fragment which could recruit ribosome or protein factors.
In the present study, expression of chimeric construct with an in-frame ORF-EGFP gene demonstrated that ATXN8OS RNA is
translatable in various human cells [70]. It indicated that the ATXN8OS putative ORF protein could be translatable and may be expressed via cap-independent IRES. We could possibly define the IRES activity of ATXN8OS to study the plausible pathogenesis of SCA8.
40
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Figure 1. Genetic modifier screening of Tau induced toxicity in Drosophila eye and notal bristle of 1-day-old adult flies. (A-U) Eye;
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(A’-U’) Notal bristle. (A) Control flies overexpressing GFP showing normal eye morphology with regular arrangement of ommatidia.
Genotype: GMR-Gal4/UAS-GFP;+/+. (A’) Normal bristle pattern in flies expressing GFP. Genotype: UAS-GFP/+; Eq-GAL4/+. (B) Expression of human Tau caused moderate rough eye phenotype. Genotype:
GMR-Gal4/+; UAS-Tau/+. (B’) Bristle loss induced by expression of Tau. Genotype: +/+; Eq-Gal4/UAS-Tau. (C-G) Selected genes involved in autophagy pathway modulate ectopic Tau induced toxicity in eye.
Genotype: GMR-Gal4/CyO; UAS-Tau/TM3 in trans to alleles indicated.
(C’-G’) Selected autophagy genes modulated Tau toxicity in the nota.
Genotype: +/+; Eq-Gal4, UAS-Tau/TM6B in trans to alleles indicated.
(H-N) Chaperones exhibited less effect on Tau induced toxicity in retina of flies. Genotype: GMR-Gal4/CyO; UAS-Tau/TM3 in trans to alleles indicated. (H’-N’) Chaperones modulated Tau toxicity in nota of flies.
Genotype: +/+; Eq-Gal4, UAS-Tau/TM6B in trans to alleles indicated.
(O and O’) Functional deficits in proteasome did not affect ectopic Tau induced toxicity in either eyes or nota. Genotype: (O) GMR-Gal4/+;
UAS-Tau/pros261. (O’) +/+; Eq-Gal4, UAS-Tau/pros261. (P and P’) Downregulation of smt3 suppressed Tau toxicity in moderate degree in both retina and nota of flies. Genotype: (P) GMR-Gal4/+;
UAS-Tau/smt304493. (P’) +/+; Eq-Gal4, UAS-Tau/ smt304493. (Q and Q’) Expression of the yu gene suppressed Tau toxicity in both eyes and nota.
Genotype: (Q) yuEP1400/+; GMR-Gal4/+; UAS-Tau/+. (Q’) yuEP1400/+;
+/+; Eq-Gal4, UAS-Tau/ +. (R and R’) Loss-of-function mutations in yu enhanced Tau toxicity in both eyes and nota. (R) yuKG02745/+;
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GMR-Gal4/+; UAS-Tau/+. (R’) yuKG02745/+; +/+; Eq-Gal4, UAS-Tau/ +.
(S and S’) Loss-of-function mutations in dbr enhanced Tau toxicity in both eyes and nota. (S) GMR-Gal4/+; UAS-Tau/dbrEP9. (S’) +/+;
Eq-Gal4, UAS-Tau/dbrEP9. (T and T’) Silencing of rpi suppressed Tau toxicity in both eyes and nota. (T) GMR-Gal4/+; UAS-Tau/UAS-rpiRNAi. (T’) +/+; Eq-Gal4, UAS-Tau/UAS-rpiRNAi. (U and U’) Overexpression of rpi has no effect on Tau induced toxicity in both eyes and nota. Genotype:
(U) GMR-Gal4/+; UAS-Tau/UAS-rpi. (U’) +/+; Eq-Gal4,
UAS-Tau/UAS-rpi. (V) Quantification of the notal bristle number of 1-day-old flies (A’-U’) assessed by student’s t-test. Each bar represents the mean ± SD of three independent experiments. ** P < 0.01; *** P <
0.001. Quantification of the bristle number as follows: (A’) 225.16±19.21, n=50; (B’) 98.67±7.91, n=55; (C’) 82.35±8.41, n=20; (D’) 89.50±6.46, n=20; (E’) 109.00±8.70, n=20; (F’) 112.80±8.35, n=20; (G’) 140.80±8.53, n=20; (H’)103.27±11.88,n=30; (I’) 96.00±8.96, n=30; (J’) 86.80±14.09, n=20; (K’) 79.80±7.19, n=20; (L’) 84.41±7.12, n=22; (M’) 121.23±
6.70,n=22; (N’) 100.81±8.66, n=21; (O’) 101.13±12.41, n=30; (P’) 124.87±13.50, n=30; (Q’) 127.35±11.05, n=20; (R’) 90.25±9.41, n=20;
(S’) 90.44±9.07, n=16; (T’) 144.70±8.24, n=20; (U’) 96.93±11.29, n=30.
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Figure 2. SEM of eye micrography and notum used in the genetic
modifier screens. (A-J) Eye; (A’-J’) Nortal bristle. Both systems gave the similar results. The notal bristle system seemed more sensitive because many modifiers were recovered by using notal bristle system but not by the eye system (Fig. 3 and Table 1). In most cases, the phenotypic changes in notal bristles were stronger and easier to be identified.
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Figure 3. Downregulation of rpi extends the lifespan and improves the motor function of tauopathy flies. (A and B) Representative notum images of 1-day-old control (A) and rpiRNAi (B) flies driven by Eq-Gal4.
Both control and rpi silencing flies shown normal bristle pattern.
Genotype: (A) Eq-Gal4/+. (B) Eq-Gal4/UAS-rpiRNAi. (C) Quantification of the bristle number of control (243.17±24.85) and rpiRNAi (240.87±22.67) driven by Eq-Gal4. The RNA interference knockdown expression of rpi has no effect itself when compared with the control flies assessed by Student’s t-test (P = 0.709). (D) In the survivorship assay, we found that the lifespan of tauopathy flies (average lifespan: 50.3 days, n=225) was significantly reduced when compared with the control flies (average lifespan: 70.8 days, n=234). Knocking down the expression of rpi
51
extended the lifespan of flies (average lifespan: 78.5 days, n=212) and significantly increased the survivorship of tauopathy flies (average lifespan: 61.4 days, n=233). The log-rank test was used for survival analysis. (E) The novel climbing ability assay was used to assess the locomotor activity of flies due to the natural negative geotaxis. The climbing index (CI) was computed as follows: CI =Σ(nm)/10, where n is the number of flies in a given scoring area, and m is the score for the given scoring area (1-5). The mobility performance of the control and downregulation rpi flies did not decline significantly over the course of 30 days. Tauopathy flies impaired motor function and CI was
significantly lower than control flies at age 15 and 30 days. Nevertheless, neuronal downregulation of rpi significantly improve the climbing ability of tauopathy flies at age of 15 and 30 days. Statistical analysis were performed using Student’s t-test (n = 100, ** P < 0.01, *** P < 0.001).
(D and E) Genotype: control: elav-Gal4/+. Tau: elav-Gal4/+;
UAS-Tau/+. rpiRNAi: elav-Gal4/+; UAS-rpiRNAi/+. Tau + rpiRNAi: elav-Gal4/+;UAS-Tau/UAS-rpiRNAi.
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Figure 4. The knockdown of rpi does not alter Tau phosphorylation. (A) A group of phosphorylation sites on Tau that are highly associated with neurodegeneration are designated as SP/TP sites. Immunoblotting experiments were using pS202, pT212, pT231, pS262, and pS396
antibodies which recognized these SP/TP sites specifically. The levels of total Tau were assessed by polyclonal antibody against Tau from Dako.
The protein blot was reprobed for β actin to document equivalent protein loading. (B) Knocking down the expression of rpi did not alter the levels of phosphor-Tau species and total Tau in the brain of 1-day-old tauopathy
53
flies assessed by Student’s t-test. Fly genotypes: control: elav-Gal4/+.
Tau: elav-Gal4/+; UAS-Tau/+. Tau + rpiRNAi: elav-Gal4/+;UAS-Tau/UAS-rpiRNAi.
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Figure 5. Neuronal downregulation of rpi increases NADPH and the reduced form of glutathione levels (GSH) in the tauopathy flies. (A) NADPH levels were determined from 1-day-old fly head extracts of the indicated genotype and normalized by its protein content. Equivalent amounts of NADPH were detected in control and tauopathy flies.
Knocking down the expression of rpi increase NADPH levels by 40%
when compared with the control or tauopathy flies. rpi knockdown resulted in a 30% increase in the levels of GSH in tauopathy flies. (B) Reduced GSH levels were determined from 1-day-old fly head extracts of the indicated genotype and normalized by its protein content. The
reduced GSH levels were significantly decreased in tauopathy flies when compared with control flies. Interesting, rpi knockdown resulted in a 1.67-fold increase in the levels of GSH in tauopathy flies. Each bar represents the mean ± SD of three independent experiments. * P < 0.05;
** P < 0.01; *** P < 0.001. Fly genotypes: control: elav-Gal4/+. Tau:
elav-Gal4/+; UAS-Tau/+. rpiRNAi: elav-Gal4/+; UAS-rpiRNAi/+. Tau + rpiRNAi: elav-Gal4/+;UAS-Tau/UAS-rpiRNAi.
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56
Figure 6. Reduced tubulin tyrosine ligase-like (TTLL) genes expressions enhance Tau-induced toxicity. (A-H) Representative notum images of control, Tau, and Tau in trans to alleles indicated from 1-day-old flies.
Notal expression of Tau causes bristal loss. Downregulation of dTTLL1, dTTLL3A, dTTLL3B, dTTLL6, dTTLL12, and dTPGS2 enhanced Tau- induced toxicity. Genotype: (A) Eq-Gal4/+. (B) Eq-Gal4/UAS-Tau. (C) dTTLL1-Ri109628/+; Eq-Gal4/UAS-Tau. (D) dTTLL3A-Ri108627/+;
Eq-Gal4/UAS-Tau. (E) dTTLL3B-Ri104449/+; Eq-Gal4/UAS-Tau. (F) dTTLL12-Ri106694/+; Eq-Gal4/UAS-Tau. (G) dTTLL6-Ri103773/+;
Eq-Gal4/UAS-Tau. (H) dTPGS2-Ri108070/+; Eq-Gal4/UAS-Tau. (I) Quantification of the bristle number of control (225.16±19.21) , Tau (98.67±7.91), and Tau in trans to dTTLL1-Ri109628 (82.20±11.76), dTTLL3A-Ri108627 (87.27±12.82), dTTLL3B-Ri104449 (86.33±12.13), dTTLL12-Ri106694 (80.80±11.77), dTTLL6-Ri103773 (78.73±11.01), dTPGS2-Ri108070 (82.83±14.12). *** P < 0.001. (J) Protein sequence alignment between dTTLL1 and hTTLL1.
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Figure 7. Overexpression of hTTLL1 attenuates Tau toxicity and extends the lifespan of tauopathy flies. (A -D) Representative notum images of control, hTTLL1, Tau, and Tau + hTTLL1 flies driven by Eq-Gal4. Both control and hTTLL1 flies shown normal bristle pattern. Bristle loss
induced by expression of Tau. Overexpression of hTTLL1 suppressed Tau toxicity. Genotype: (A) Eq-Gal4/+. (B) hTTLL1/+; Eq-Gal4/+. (C)
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Eq-Gal4/UAS-Tau. (D) hTTLL1/+; Eq-Gal4/UAS-Tau. (E) Quantification of the bristle number of control (234.17±19.70) , hTTLL1 (239.60±28.38) , Tau (136.53±10.93), Tau in trans to hTTLL1 (176.73±16.92) flies driven by Eq-Gal4. The overexpression of hTTLL1 has no effect itself when compared with the control flies assessed by Student’s t-test (P = 0.709).
The overexpression of hTTLL1 significantly increased the bristle number
The overexpression of hTTLL1 significantly increased the bristle number