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  • Results from experiments in model systems have suggested tha

    2021-07-27

    Results from experiments in model systems have suggested that the DPRs translated from the HRE of C9ORF72 are toxic and play a role in disease pathogenesis (reviewed in Taylor et al. (2016)). Although the DPRs are present in neurons and glia in various regions of the central nervous system (CNS) early in disease (Freibaum and Taylor, 2017), they surprisingly tend to be more frequent in CNS regions that have less pathology, such as the cerebellum, rather than the main targeted tissue – namely the motor system (MacKenzie et al., 2014). GA is the most common DPR found in autopsy tissue, and also the most aggregate-prone and amyloidogenic (reviewed in (Freibaum and Taylor, 2017). The mechanism for toxicity remains unclear, but might relate to its aggregate formation. PR and GR are thought to be the most toxic DPRs (reviewed in Freibaum and Taylor (2017)). GP can be detected in the cerebrospinal fluid of patients with C9ORF72-associated FALS as well as asymptomatic carriers of the HRE (Gendron et al., 2017); however, the levels of GP are relatively stable over time, showing no fluctuation related to disease progression. Of note, GR is recruited by GA into cytoplasmic inclusions (Yang et al., 2015). PA and PR are the least abundant, perhaps because they are derived from the antisense transcript or perhaps because the Pyocyanin australia used for their detection have suboptimal sensitivity. In the present study we investigated translation of DPRs following transfection of cells with a construct that encoded 75 repeats of C9ORF72 and flanking sequence. We found that translation initiation of GA occurred from a CUG codon upstream from the repeat. Interestingly, translation of GA involved internal ribosome entry and partly depended on eIF2A, a non-canonical initiation factor that is used in translation by certain viruses (Kim et al., 2011) and by cells experiencing various stress conditions (Sendoel et al., 2017; Starck et al., 2012). Furthermore, translation of GA was associated with induction of the integrated stress response (ISR), while the ISR led to continuing GA translation, suggesting that the interaction of GA translation and the ISR leads to a self-sustaining pathogenic cycle.
    Materials and methods
    Results
    Discussion The identification of the C9ORF72 G4C2 HRE as the most common genetic cause of FALS, FTD, and ALS-FTD as well as the cause of 5–10% of sporadic ALS was a sentinel finding that has raised a number of questions, many of which remain unanswered. Several mechanisms by which this repeat expansion cause disease have been proposed including toxicity from DPRs translated from the HREs. Although evidence of toxicity from the DPRs comes from varied experimental models, it still remains controversial. Although the C9ORF72 HRE is in a non-coding region with respect to the main gene product, DPRs are synthesized from the repeat from both the positive and negative strand of mRNA. DPRs are an authentic product of translation of C9ORF72 HREs because they are present in neural cells in autopsy tissue from patients with this mutation. This unconventional translation, known as RAN translation, can occur in a number of expanded repeat diseases from repeats in both the non-coding and coding region of the main gene product. For example, translation of DPRs from the CAG repeat in the coding region of huntingtin mRNA occurs in addition to translation of polyglutamines in huntingtin (Bañez-Coronel et al., 2015). Unconventional cap-independent translation has also been increasingly found in cellular genes, frequently from an uORF or IRES (Gebauer and Hentze, 2016; Hinnebusch et al., 2016; Weingarten-Gabbay et al., 2016). Transfection of constructs containing 75 G4C2 repeats with 113 nucleotides 5′ to the repeats and 99 nucleotides 3′ to the repeats showed that GA can be translated in both HEK293 and NSC34 cells as well as chick embryo spinal cord neural cells. A CUG with a good Kozak consensus sequence upstream of the HRE provided a potential candidate for an initiation codon, especially since this codon has been recognized as a major alternative translation start site for uORFs in cells (Starck et al., 2012). Of note, the Kozak sequence may have a greater influence on non-AUG initiation codons than AUG initiation codons (Kearse and Wilusz, 2017). Other studies have recently shown that this upstream CUG is involved in GA translation in cultured cells (Green et al., 2017; Tabet et al., 2018). Our mutagenesis studies demonstrated that this upstream CUG was indeed the GA translation initiation site both in cultured cells as well as in vivo in chick embryo spinal cord neural cells. The HRE was translated in cultured cells as well as chick embryo spinal cord neural cells when placed as the second cistron in a bicistronic construct, suggesting that this translation occurred by means of an IRES and was independent of the mRNA 5′-end. The location of a translation initiation site upstream of a repeat expansion has also been described with the CGG expanded repeats in FMR1 (Sellier et al., 2017).