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CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.

Williams KL, Topp S, Yang S, Smith B, Fifita JA, Warraich ST, Zhang KY, Farrawell N, Vance C, Hu X, Chesi A, Leblond CS, Lee A, Rayner SL, Sundaramoorthy V, Dobson-Stone C, Molloy MP, van Blitterswijk M, Dickson DW, Petersen RC, Graff-Radford NR, Boeve BF, Murray ME, Pottier C, Don E, Winnick C, McCann EP, Hogan A, Daoud H, Levert A, Dion PA, Mitsui J, Ishiura H, Takahashi Y, Goto J, Kost J, Gellera C, Gkazi AS, Miller J, Stockton J, Brooks WS, Boundy K, Polak M, Muñoz-Blanco JL, Esteban-Pérez J, Rábano A, Hardiman O, Morrison KE, Ticozzi N, Silani V, de Belleroche J, Glass JD, Kwok JB, Guillemin GJ, Chung RS, Tsuji S, Brown RH, García-Redondo A, Rademakers R, Landers JE, Gitler AD, Rouleau GA, Cole NJ, Yerbury JJ, Atkin JD, Shaw CE, Nicholson GA, Blair IP - Nat Commun (2016)

Bottom Line: CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)).Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate.This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

No MeSH data available.


Related in: MedlinePlus

Mutant cyclin F impairs ubiquitin-mediated proteasomal degradation.NSC-34 cells were co-transfected with GFPu and either wild type or mutant cyclin F, tagged with mCherry. GFPu fluorescence intensity was analysed by flow cytometry 48 h post transfection. (a) Plot of GFPu fluorescence intensity following flow cytometry. A significantly higher level of GFPu fluorescence was observed in cells expressing novel cyclin F mutations (blue data points) when compared with those expressing wt CCNF (red data points) WT v FALS/FTD P=0.0017, d.f.=11; WT v SALS/FTD P=0.001, d.f.=7; two-tailed unpaired Student's t-test). (b) The higher level of GFPu fluorescence was independent of the level of cyclin F as quantified using mCherry signal—R-squared=0.13. Red dashed lines represent the WT mean. Data are represented as mean,±s.e.m. n=3 (n is one experiment consisting of the mean of 50,000 cells); **P<0.01. d.f., degrees of freedom.
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f2: Mutant cyclin F impairs ubiquitin-mediated proteasomal degradation.NSC-34 cells were co-transfected with GFPu and either wild type or mutant cyclin F, tagged with mCherry. GFPu fluorescence intensity was analysed by flow cytometry 48 h post transfection. (a) Plot of GFPu fluorescence intensity following flow cytometry. A significantly higher level of GFPu fluorescence was observed in cells expressing novel cyclin F mutations (blue data points) when compared with those expressing wt CCNF (red data points) WT v FALS/FTD P=0.0017, d.f.=11; WT v SALS/FTD P=0.001, d.f.=7; two-tailed unpaired Student's t-test). (b) The higher level of GFPu fluorescence was independent of the level of cyclin F as quantified using mCherry signal—R-squared=0.13. Red dashed lines represent the WT mean. Data are represented as mean,±s.e.m. n=3 (n is one experiment consisting of the mean of 50,000 cells); **P<0.01. d.f., degrees of freedom.

Mentions: Aberrant misfolded proteins are targeted for disposal by protein degradation pathways including the UPS and autophagic–lysosomal system, both of which are components of the complex network that maintains protein homeostasis (proteostasis). The accumulation of neuronal protein aggregates in ALS patients implicates dysfunction of the proteostasis network through inappropriate or inadequate response to aberrant proteins17. As a ubiquitin–protein ligase, cyclin F catalyses the transfer of activated ubiquitin to target proteins15. To investigate whether the ALS/FTD-associated variants in cyclin F lead to proteostasis dysfunction, we used the UPS reporter, GFPu, that consists of a 16 amino-acid degron (CL1, a specific substrate for the UPS), fused to the carboxyl terminus of green fluorescent protein (GFP)18. The degron sequence ensures rapid degradation of the GFP fusion through ubiquitin-mediated pathways, and the accumulation of this GFP reporter indicates UPS impairment. We confirmed that GFPu signal correlates with UPS function in a motor neuron-like cell line (NSC-34) by inhibiting proteasome function using either the chemical inhibitor MG132, or expression of a mutant huntingtin exon 1 fragment containing an expanded polyQ sequence previously shown to inhibit the proteasome19 (Supplementary Fig. 2). Next, we co-transfected the NSC-34 cell line with GFPu and either mutant cyclin F or wild-type cyclin F (Supplementary Table 4). Significantly higher levels of GFPu fluorescence were observed for cyclin F with ALS/FTD-associated variants, indicating UPS dysfunction (Fig. 2a). This effect was independent of cyclin F expression levels (Fig. 2b). To examine whether the significant accumulation of GFPu arose from the loss of proteasome activity or occurred upstream of the proteasome, we used two separate ubiquitin-independent small peptide 20S proteasome activity assays. These demonstrated that the UPS dysfunction was not due to altered proteolysis in the proteasome (Supplementary Fig. 3), consistent instead with the dysfunction stemming from abnormal ubiquitination or transport to the proteasome, mechanisms that are mediated, in part, by cyclin F. Consistent with these observations, western blotting confirmed the presence of significantly more ubiquitinated proteins in neuronal cell lines expressing mutant cyclin F (Fig. 3a,b). Collectively our data suggest that ALS/FTD-associated variants in CCNF modify the activity of SCFCyclin F resulting in overaccumulation of ubiquitinated proteins. To specifically demonstrate this, we found higher levels of the known SCFCyclin F target, RRM2 (Fig. 3a,b), including higher levels of ubiquitinated RRM2 (Fig. 3c), in neuronal cells expressing mutant cyclin F. Notably, we also observed substantially elevated levels of ubiquitinated TDP-43 in neuronal cells expressing mutant cyclin F (Fig. 3c).


CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.

Williams KL, Topp S, Yang S, Smith B, Fifita JA, Warraich ST, Zhang KY, Farrawell N, Vance C, Hu X, Chesi A, Leblond CS, Lee A, Rayner SL, Sundaramoorthy V, Dobson-Stone C, Molloy MP, van Blitterswijk M, Dickson DW, Petersen RC, Graff-Radford NR, Boeve BF, Murray ME, Pottier C, Don E, Winnick C, McCann EP, Hogan A, Daoud H, Levert A, Dion PA, Mitsui J, Ishiura H, Takahashi Y, Goto J, Kost J, Gellera C, Gkazi AS, Miller J, Stockton J, Brooks WS, Boundy K, Polak M, Muñoz-Blanco JL, Esteban-Pérez J, Rábano A, Hardiman O, Morrison KE, Ticozzi N, Silani V, de Belleroche J, Glass JD, Kwok JB, Guillemin GJ, Chung RS, Tsuji S, Brown RH, García-Redondo A, Rademakers R, Landers JE, Gitler AD, Rouleau GA, Cole NJ, Yerbury JJ, Atkin JD, Shaw CE, Nicholson GA, Blair IP - Nat Commun (2016)

Mutant cyclin F impairs ubiquitin-mediated proteasomal degradation.NSC-34 cells were co-transfected with GFPu and either wild type or mutant cyclin F, tagged with mCherry. GFPu fluorescence intensity was analysed by flow cytometry 48 h post transfection. (a) Plot of GFPu fluorescence intensity following flow cytometry. A significantly higher level of GFPu fluorescence was observed in cells expressing novel cyclin F mutations (blue data points) when compared with those expressing wt CCNF (red data points) WT v FALS/FTD P=0.0017, d.f.=11; WT v SALS/FTD P=0.001, d.f.=7; two-tailed unpaired Student's t-test). (b) The higher level of GFPu fluorescence was independent of the level of cyclin F as quantified using mCherry signal—R-squared=0.13. Red dashed lines represent the WT mean. Data are represented as mean,±s.e.m. n=3 (n is one experiment consisting of the mean of 50,000 cells); **P<0.01. d.f., degrees of freedom.
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Related In: Results  -  Collection

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f2: Mutant cyclin F impairs ubiquitin-mediated proteasomal degradation.NSC-34 cells were co-transfected with GFPu and either wild type or mutant cyclin F, tagged with mCherry. GFPu fluorescence intensity was analysed by flow cytometry 48 h post transfection. (a) Plot of GFPu fluorescence intensity following flow cytometry. A significantly higher level of GFPu fluorescence was observed in cells expressing novel cyclin F mutations (blue data points) when compared with those expressing wt CCNF (red data points) WT v FALS/FTD P=0.0017, d.f.=11; WT v SALS/FTD P=0.001, d.f.=7; two-tailed unpaired Student's t-test). (b) The higher level of GFPu fluorescence was independent of the level of cyclin F as quantified using mCherry signal—R-squared=0.13. Red dashed lines represent the WT mean. Data are represented as mean,±s.e.m. n=3 (n is one experiment consisting of the mean of 50,000 cells); **P<0.01. d.f., degrees of freedom.
Mentions: Aberrant misfolded proteins are targeted for disposal by protein degradation pathways including the UPS and autophagic–lysosomal system, both of which are components of the complex network that maintains protein homeostasis (proteostasis). The accumulation of neuronal protein aggregates in ALS patients implicates dysfunction of the proteostasis network through inappropriate or inadequate response to aberrant proteins17. As a ubiquitin–protein ligase, cyclin F catalyses the transfer of activated ubiquitin to target proteins15. To investigate whether the ALS/FTD-associated variants in cyclin F lead to proteostasis dysfunction, we used the UPS reporter, GFPu, that consists of a 16 amino-acid degron (CL1, a specific substrate for the UPS), fused to the carboxyl terminus of green fluorescent protein (GFP)18. The degron sequence ensures rapid degradation of the GFP fusion through ubiquitin-mediated pathways, and the accumulation of this GFP reporter indicates UPS impairment. We confirmed that GFPu signal correlates with UPS function in a motor neuron-like cell line (NSC-34) by inhibiting proteasome function using either the chemical inhibitor MG132, or expression of a mutant huntingtin exon 1 fragment containing an expanded polyQ sequence previously shown to inhibit the proteasome19 (Supplementary Fig. 2). Next, we co-transfected the NSC-34 cell line with GFPu and either mutant cyclin F or wild-type cyclin F (Supplementary Table 4). Significantly higher levels of GFPu fluorescence were observed for cyclin F with ALS/FTD-associated variants, indicating UPS dysfunction (Fig. 2a). This effect was independent of cyclin F expression levels (Fig. 2b). To examine whether the significant accumulation of GFPu arose from the loss of proteasome activity or occurred upstream of the proteasome, we used two separate ubiquitin-independent small peptide 20S proteasome activity assays. These demonstrated that the UPS dysfunction was not due to altered proteolysis in the proteasome (Supplementary Fig. 3), consistent instead with the dysfunction stemming from abnormal ubiquitination or transport to the proteasome, mechanisms that are mediated, in part, by cyclin F. Consistent with these observations, western blotting confirmed the presence of significantly more ubiquitinated proteins in neuronal cell lines expressing mutant cyclin F (Fig. 3a,b). Collectively our data suggest that ALS/FTD-associated variants in CCNF modify the activity of SCFCyclin F resulting in overaccumulation of ubiquitinated proteins. To specifically demonstrate this, we found higher levels of the known SCFCyclin F target, RRM2 (Fig. 3a,b), including higher levels of ubiquitinated RRM2 (Fig. 3c), in neuronal cells expressing mutant cyclin F. Notably, we also observed substantially elevated levels of ubiquitinated TDP-43 in neuronal cells expressing mutant cyclin F (Fig. 3c).

Bottom Line: CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)).Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate.This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

No MeSH data available.


Related in: MedlinePlus