This study demonstrates that ALS/FTD-associated mutations in cyclin F perturb ER homeostasis by inhibiting two key functions; ERAD and transport of proteins from the ER to the Golgi apparatus. Consistent with these observations, Golgi fragmentation, ER stress, and induction of apoptosis was also detected in cell lines and mouse cortical primary neurons expressing mutant cyclin F. Together these data imply that dysfunction to the ER is an important process in neuronal degeneration induced by mutant cyclin F, thus providing novel insights into CCNF-associated ALS/FTD.
ER-Golgi transport is a vital gateway to the endomembrane system because one third of all proteins transit through the ER-Golgi compartments before reaching their final cellular locations [18, 57]. Our results imply that ALS/FTD-associated p.S621G and p.S195R mutations in cyclin F perturb the first stage of ER-Golgi transport: the budding of COPII vesicles from the ER. The COPII coat is composed of five separate proteins, including Sec23 and Sec31, and its assembly is essential for the formation of transport vesicles on the cytosolic face of the ER membrane. Curvature of the ER membrane, concentration of cargo and vesicular release then results. Defective COPII vesicles are known to inhibit secretion [40] and here we detected several abnormalities in COPII in mutant cyclin F cells. Less COPII and less vesicular cargo were present in ER-derived membranes obtained from mutant cyclin F expressing cells compared to controls. Furthermore, the Sec31-clusters (representing one or more ER-exit sites) were smaller in these cells, implying that either fewer COPII vesicles were formed overall or that the same number of COPII vesicles were present, but each contained less Sec31. Typical COPII vesicles are 60–70 nm in diameter, and it was not possible to resolve individual COPII vesicles or ER-exit sites using the methods used here. Furthermore, whilst Sec23 levels correlate with the load of protein cargo [40], here the levels of Sec23 were comparable to cargo load, suggesting that the defect is related to aberrant COPII vesicles themselves rather than the incorporation of protein cargo. Hence, these results imply that there is a defect in the formation of COPII vesicles and/or ER-exit sites in mutant cyclin F expressing cells.
COPII vesicles are normally too small to transport some secreted macromolecules, including procollagen fibrils, pre-chylomicrons, and pre- very low-density lipoproteins (VLDLs). However, ubiquitination of Sec31 by the CUL3-KLHL12 complex allows for the generation of large vesicles to facilitate the transport of these bulky cargo, thus controlling the traffic of COPII vesicles [40, 58]. Calcium-dependant control of CUL3-KLHL12 also ubiquitinates lysine residues on Sec31 [40, 59]. Similarly, deubiquitylation of Sec31 by USP8 antagonizes the formation of large procollagen-containing carriers [60]. The exact site of Sec31 ubiquitination may not be crucial for regulation of COPII vesicle trafficking, but it is a signal to recruit other effectors to assemble the coat or to regulate its catalytic activity. We previously demonstrated that mutant cyclin FS621G dysregulates ubiquitination at Lys48, resulting in disruption of biological networks responsive for cellular survival and maintenance [11, 13]. Here, we demonstrate that in cells expressing mutant cyclin F, ubiquitination of Sec31 was reduced and bulk secretion of large proteins (> 100kDa) was inhibited, although the secretion of proteins smaller than this was unchanged. This together with inhibition of ER-Golgi trafficking in these cells implies that aberrant ubiquitination of Sec31 impairs the formation of COPII vesicles, forming smaller ER-exit site clusters and inhibiting the transport of large cargoes, which would ultimately impact neuronal functions. Future studies are therefore warranted to confirm this possibility and to identify the specific large cargoes whose secretion is inhibited by mutant cyclin F. Mutations in the genes encoding Sec23A [61] or Sar1B [62] lead to the accumulation of large proteins such as collagen, or lipid particles into the ER [61] which causes Skull-Lenticular-Sutural Dysplasia [61] and Chylomicron Retention Disease [62]. This highlights the importance of the integrity of COPII vesicles in the regulation of protein secretion.
Collagen forms the major structural component of the extracellular matrix and it is the most abundant cargo for COPII vesicles [60]. This is not surprising given that collagen constitutes up to 30% of the total protein mass of a typical organism. It was previously believed that fibrillar collagens are absent in the brain [63]. However, neurons are now known to express collagen [64, 65], and an increasing number of studies highlight key roles for collagen in both the PNS and CNS [66]. Whilst this area remains poorly understood, it is clear that expression and deposition of collagen in the nervous system is highly dynamic and tightly controlled, and is implicated in regulation of axonal outgrowth and synaptic differentiation [67]. Furthermore, some types of collagen are regulated in response to stress and are dysregulation in several neurological conditions, including Alzheimer’s diseases[66]. regulated in conditions. COL6
Ubiquitination also regulates ERAD because poly-ubiquitin chains on ERAD substrates are remodelled at several stages during ERAD [68, 69]. Interestingly, we also detected impairment of ERAD in cells expressing mutant cyclin F. ERAD normally removes terminally misfolded proteins from the ER lumen or membrane, targeting them for degradation to membrane-embedded E3 ligase complexes, where they undergo ubiquitination on their cytosolically exposed protein domains. Hence, misfolded proteins in the ER would not be degraded efficiently in cyclin F-associated ALS/FTD, leading to their accumulation in the ER. This would further induce ER stress and disrupt ER homeostasis. These mechanisms may all combine, thus exacerbating ER stress and further perturbing ER homeostasis. Consistent with activation of pro-apoptotic CHOP during ER stress, we also demonstrate that expression of ALS/FTD cyclin F mutants induces neuronal death. This finding is also consistent with apoptotic death observed in the transient fish models overexpressing S621G cyclin F [13].
Impairment of ER-Golgi and other forms of transport has been previously described in ALS [23, 70–73], in cells expressing mutant SOD1 [23], mutant TDP-43 [23], mutant FUS [23] or mutant ubiquilin2 [31], and these events were previously linked to ER stress [23]. ER stress has now been widely implicated in ALS and mutations in SOD1 [44], TARDBP [74], FUS [75], OPTN [76–78], VCP [79], UBQLN2 [31], VAPB [80, 81] and C9orf72 [82] all induce ER stress. Previously it has been established that mutant forms of SOD1 and TDP-43 induce ER stress from the cytoplasm [70, 71, 83], or from the cytoplasmic face of the ER [23] respectively, by inhibiting ER-Golgi transport [23]. Preliminary experiments examining whether cyclin F is present in the ER were inconclusive, raising the question of how ER stress is induced in ALS/FTD. As ALS mutant cyclin F mislocalizes to the cytoplasm where it promotes cytoplasmic aggregation of TDP-43 [84], it is possible that mutant cyclin F induces ER stress from the cytoplasm by inhibiting ER-Golgi transport. However, we cannot rule out the possibility that mutant cyclin F may directly induce ER stress from the ER itself, although mutant cyclin F aberrantly ubiquitinates Sec 31, which is also localised in the cytoplasm.
Golgi fragmentation is also a well-described event in ALS [24, 53, 85] where the Golgi apparatus undergoes morphological changes, resulting in disruption of its characteristic ribbon-like structure [85, 86]. Fragmentation of the Golgi apparatus has been described in sporadic ALS patients [87] and before disease onset in SOD1G93A mice [88], prior to neuromuscular denervation and axon retraction [89]. It is also a feature of other neurodegenerative diseases [85] but it has not been previously described for mutant cyclin F. In this study, we demonstrate that ALS/FTD-associated mutations in cyclin F trigger Golgi fragmentation. The proper organization of the Golgi depends on efficient bidirectional ER-Golgi vesicle transport [86, 90] and both membrane flow and cargo load influence its structure and function. Hence, blocking the export of cargo-containing ER carriers [91, 92] or depleting cargo receptors [93] results in Golgi fragmentation and some of the tubulo-vesicular Golgi clusters can further fuse with the ER, increasing ER stress [94]. In addition, inhibition of intra-Golgi trafficking or vesicle transport from the Golgi to the plasma membrane can result in Golgi fragmentation, if prolonged [50–52]. Hence, in this study, it is possible that the inhibition of ER-Golgi transport by mutant cyclin F triggers Golgi fragmentation and ER stress. However, there are many possible cellular stressors than induce Golgi fragmentation so the directionality of these links cannot be conclusively established.