Repeat-associated non-ATG initiated (RAN) translation is a form of polypeptide synthesis which does not depend on an AUG codon. RAN-translation has been demonstrated in other repeat-associated diseases such as fragile X-associated tremor ataxia syndrome (FXTAS) and fragile X syndrome (FXS): A CGG repeat expansion in the 5’ UTR of the fragile X mental retardation gene (FMR1) causes the translation of poly-Gly or poly-Ala, depending on the reading frame used by the ribosome. The size of this expansion determines whether the carrier will be more prone to develop FXTAS or FXS.
FXS is associated with what is known as a full mutation, meaning that the expansion contains over 200 repeats (Yu et al., 1991, Oberle et al., 1991). However, carriers of the premutation, consisting of 55-200 repeats, are more prone to develop FXTAS, and the penetrance will be dependent on age and repeat size (Jacquemont et al., 2004). Initiation of RAN translation of the FMR1 expansion seems to occur at a near-cognate ACG codon upstream the CGG repeats (Sellier et al., 2017). RAN-translation of the C9ALS-associated GGGGCC expansion also causes the synthesis of sense and antisense insoluble dipeptide
32 repeats (DPR) which aggregate in neurones and glia in the brain of both C9ALS and C9FTD patients (Mori et al., 2013a, Mori et al., 2013c, Ash et al., 2013, Zu et al., 2013).
The protein sequence of the DPR depends on the reading frame from which it is translated, which gives rise to a total of 5 different DPR (figure 1.3). These are –in descending order of the frequency in which they are found aggregated in the brain- poly-GA, poly-GP, poly-GR, poly-PA and poly-PR. From those, arginine-containing DPR (poly-GR and poly-PR) have been shown to be the most neurotoxic when aggregated after overexpression in cultured neurones, yeast, and drosophila. Arg-rich DPR have been mainly found to localise within the nucleolus. They interact with nuclear and nucleolar proteins, inducing nucleolar stress, which causes impairment of normal gene expression, including the dysregulation and silencing of mRNAs (Tao et al., 2015).
Nucleolar stress is usually denoted by the cytoplasmic translocation of B23 (Yao et al., 2010), which is responsible for ribosome biogenesis, promotes cell survival and regulates cell cycle progression (Lee et al., 2008, Lindstrom, 2011). Arg-rich DPR aggregation in the nucleus also causes the enlargement and dysfunction of the nucleolus in neurones (Mizielinska et al., 2017). The relevance of poly-GR aggregation seems more relevant than that of poly-PR because while poly-GR is found frequently in post mortem ALS/FTD brains, poly-PR is rarely found (Mackenzie et al., 2015, Schludi et al., 2015).
Surprisingly, poly-GA, the most commonly found form of DPR, appears to reduce the toxicity caused by arginine-rich DPR in human induced-pluripotent stem cell-derived neurones and drosophila. Poly-GA (15 and 50 repeats) was also found to form toxic fibrils in solution and to cause motor deficits in mice when expressed using an adeno associated virus (AAV) viral system injected in the cerebral ventricles. It remains to be ascertained whether the level of expression reached using viral vectors is similar to that of human C9ALS and whether it produces neurotoxicity at normal pathological levels (as opposed to overexpression levels) in the human brain.
It would be expected to find correlations between neuronal death or disease severity and the degree of DPR accumulation if DPR were indeed the main cause of C9ALS/FTD
33 pathogenesis. However, even with DPR being prevalent in some key areas of these patients’ brains (Mackenzie et al., 2013), the relative infrequency of the most toxic, Arg-rich DPR species, suggests that they could merely be pathological hallmarks in some regions of the brain, but be of no neurotoxic consequence. Opposing this view, there is also the possibility that most of the neurones and glial cells containing these aggregates die before the patient does, causing ALS/FTD phenotypes and rendering neuropathologists unable to detect them post mortem.
Figure 1.3: Dipeptide repeat (DPR) sequences translated from C9orf72 expansions. GGGGCC repeats are RAN-translated into toxic polypeptides consisting in the repetition of two amino acid residues. The translated DPR depends on which DNA strand is used as the template and on the reading frame. Two of these sequences are redundant (poly-GP), so only five different DPR can result from this form of translation.
34 1.4.6 Nucleocytoplasmic transport and the C9orf72 expansion
Common ground for the mRNA and DPR toxic gain of function hypotheses seems to be the impairment of nucleocytoplasmic transport. As stated above, the most commonly aggregated protein in ALS cytoplasmic inclusions, TDP-43, is constantly shuttled between the nucleus and the cytoplasm, and so do other proteins and molecules. This transport requires GTP-bound RAs-related nuclear protein (RanGTP) to hydrolyse its bound GTP to GDP, forming RanGDP. Otherwise, importins would be inhibited in the cytoplasm, disabling cytoplasm-to-nucleus transport. Ran GTPase-activating protein (RanGAP) is essential to activate the catalytic function of RanGTP. The cytoplasmic accumulation and dysfunction of RanGAP and RanGTP, and their co-localisation with expanded C9orf72 mRNA foci have been demonstrated in C9ALS brain tissue as well as in patient iPSC-derived neurones and Drosophila models harbouring expanded C9orf72 (Zhang et al., 2015). Also, the severity of RanGTP mislocalisation correlates with the severity of TDP-43 nuclear depletion (Winton et al., 2008), meaning that TDP-43 keeps being transported out of the nucleus without making its way back in. Also, nuclear depletion of TDP-43 aggregation seems to accumulate in cells with expanded C9orf72 mRNA foci (Cooper-Knock et al., 2015). This all seems to indicate that expanded C9orf72 mRNA binds and sequesters some essential factors for the active transport of macromolecules between the nucleus and the cytoplasm. However, there is also evidence that RAN-translated poly-GA DPR can cause mislocalisation of RanGAP and nuclear envelope proteins just by itself (Zhang et al., 2016b). Cytoplasmic poly-GA also inhibits the nuclear import of transcription factor p65 and promotes the cytoplasmic aggregation of TDP-43 in neurones (Khosravi et al., 2017). This phenotype can be rescued by inducing the overexpression of importin-alpha and nuclear pore components. Furthermore, directing poly-PA to the nucleus avoids TDP-43 mislocalisation to the cytoplasm (Khosravi et al., 2017). Another study showed a lower import rate in the nucleus caused by poly-PR and poly-GR (arginine-rich) using NSC-34 cells (Shani et al., 2017). Therefore, cytoplasmic aggregation of poly-GA and expanded C9orf72 mRNA both contribute to ALS pathogenesis by disrupting nucleocytoplasmic transport.
35 1.4.7 Pathology in C9orf72 expansion-carrying patients
Surprisingly, ALS cases harbouring C9orf72 mutations show a pathological pattern which seems qualitatively very similar (but quantitatively greater) from patients not harbouring the expansion. Additionally, as described above, RAN-translated DPR also aggregate in C9ALS to form cytoplasmic and intranuclear neuronal inclusions which are negative for TDP-43, but positive for proteins related to the proteasome such as p62 and ubiquitin (Mackenzie et al., 2013, Zu et al., 2013, Mori et al., 2013c, Gendron et al., 2013, Ash et al., 2013). This neuronal aggregation accompanied by dystrophic neurites is extended in the cortex from the prefrontal cortex all the way to the visual cortex in the occipital lobe. The hippocampus is an extramotor area where TDP-43 and OPTN are usually good markers for protein inclusion pathology at stage 4, showing little involvement in the rest of the stages. However, neither of them can pick up all the abundant intra- and extra-nuclear neuronal inclusions which are labelled for p62 and DPR in granular and Purkinje cells of the cerebellum and the pyramidal neurones of the hippocampus found in C9ALS (Cooper-Knock et al., 2012, Mackenzie et al., 2014, Ash et al., 2013).
36 1.5 Oligodendrocytes and their dysfunction in ALS
There are four main types of glial cell in the adult CNS: microglia, astrocytes, ependymal cells, and oligodendrocytes. The latter are responsible for forming the myelin sheaths which wrap the neuronal axons and make them deliver the nerve impulse considerably faster than unsheathed axons. It has been shown that many ALS cases and SOD1 (G93A) miceshow demyelination of motor fibres (Verstraete et al., 2014, Ahdab et al., 2013, Kang et al., 2013, Borisow et al., 2013, Nishijima et al., 2012). Furthermore, myelin degeneration has been seen in higher mammal models of ALS. For example, canine degenerative myelopathy (CDM) is a condition that affects dogs with a naturally—
occurring mutation in their SOD1 homologue gene (Awano et al., 2009). CMD is considered a model of human ALS because affected dogs show the same type of spinal lesions and the adult-onset progressive loss of motor function (Nardone et al., 2016).
One of the main pathological features of CDM is the evidence of demyelination in the spinal cord funiculi, especially in the dorsal area of the lateral funiculi, where the lateral motor tracts are located (Griffiths and Duncan, 1975). Additionally, a transgenic line of pigs expressing ALS-causing mutant human G93A SOD1 has been made (Yang et al., 2014). These pigs showed myelin sheath degeneration in the spinal cord along with all the other common features of ALS -e.g. muscle wasting, motor disfunction and SOD1 neuronal and glial inclusions. Demyelination in these models and in human ALS patients can be caused by a number of factors, including a lack of myelin production, a lack of the appropriate receptors which allow oligodendrocyte processes to attach to the axon or other layers, or an inefficacy of the oligodendrocytes to produce competent processes to wrap the axons.