Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G₄C₂ repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G₄C₂ RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Structural and biochemical characterisation of the C9orf72-SMCR8 complex sheds light on its overall architecture and highlights its role as a multi-functional scaffold for coordinating autophagy.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin‐2 to induce motor neuron dysfunction and cell death

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS‐FTD). Ataxin‐2 with intermediate length of polyglutamine expansions (Ataxin‐2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP‐43 and P62 proteins, which are histopathological hallmarks of ALS‐FTD. SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin‐2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin‐2 toxicity, suggesting a double‐hit pathological mechanism in ALS‐FTD.     

Lost & found: C9ORF72 and the autophagy pathway in ALS/FTD

C9ORF72 expression is reduced in a substantial number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which may contribute to disease pathogenesis. However, its normal molecular function remains unknown. In this issue of The EMBO Journal, Sellier et al ( 2016 ) identified a novel protein complex consisting of C9ORF72, WDR41, and SMCR8 that acts as a GDP‐GTP exchange factor (GEF) for RAB8a and RAB39b and is regulated by TBK1, whose partial loss of function also causes ALS and FTD. They further reveal a potential modulatory role for this novel complex in macroautophagy (autophagy), especially in the context of ataxin‐2 toxicity.     

C9orf72: At the intersection of lysosome cell biology and neurodegenerative disease

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith‐Magenis chromosome region 8 (SMCR8) and WD repeat‐containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72‐containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.      

Systemic deregulation of autophagy upon loss of ALS- and FTD-linked C9orf72

A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient‐derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD‐associated p62 pathology.     

WDR72 models of structure and function: A stage-specific regulator of enamel mineralization

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72^−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72^−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72^−/− ameloblasts with retained proteins in the enamel matrix during maturation stage. H⁺/Cl⁻ exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72^−/− maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72^−/− mice as compared to wildtype controls. Moreover, Wdr72^−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Can ALS-Associated C9orf72 Repeat Expansions Be Diagnosed on a Blood DNA Test Alone?

Gene mutations that preferentially affect the CNS have been implicated in a number of neurological disorders. This leads to the possibility that a disease-causing mutation present only in CNS tissues could be missed if it were tested in a blood DNA sample only. The commonest mutation in amyotrophic lateral sclerosis (ALS) is an expansion of the hexanucleotide repeats of C9orf72. To find out if CNS-specific mutations of this gene could cause some cases of ALS we looked for differences in the size of C9orf72 repeats between DNA from the CNS and from white blood cells (WBCs) of 38 sporadic ALS patients, using a repeat-primed PCR screening test. We also looked for differences in C9orf72 repeats in WBC DNA from 6 ALS-discordant and 1 ALS-concordant monozygotic twins. Abnormally expanded C9orf72 repeats were found in 13% of the ALS CNS samples, as well as in their paired WBC DNA. The 87% of ALS CNS samples with normal-sized C9orf72 repeats had the same number of repeats in paired WBC samples. All ALS-discordant twins had the same normal numbers of WBC C9orf72 repeats. Although previous work suggests some tissue mosaicism in C9orf72 repeat size is probably present, this study indicates that this is not likely to be of sufficient magnitude to result in a normal C9orf72 repeat length in blood but an abnormally expanded repeat length in the CNS. This suggests that a blood DNA test alone will usually be sufficient to make a diagnosis of C9orf72 repeat-related ALS.     

Live-cell imaging of Aspergillus nidulans autophagy

We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This ‘autophagosome cycle’ gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS^RAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS^RAB7 or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA^RAB5/RabB^RAB5 (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE₂-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO^RAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO^RAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO^RAB1 are dissociable by mutation: rabO^A136D hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.     

Soil aggregate and particulate C and N under corn rotations: responses to management and correlations with yield

Background

Soil aggregate and particulate organic matter (POM) C and N provide valuable insight into C cycling and storage, and are sensitive to management, but effect of these pools on corn yield is unknown.

Methods

Corn yield, N uptake, and aggregate and POM C and N at 0–5, 5–25 and 25–50 cm were measured and correlated in continuous corn (Zea mays L.) (CC), strip-till corn/soybean [Glycine max (L.) Merr.] (CS), and organically managed corn/soybean/wheat (Triticum aestivum L.) with green manure (CSW).

Results

The POM differed only at 0–5 cm, where greater POM mass was found in CS than CC and CSW. Lower POM-C and POM-N was found in CSW than CC and CS. Overall, CSW had fewer macroaggregates (>250 μm) and associated C and N than CC and CS, but free silt and clay (<53 μm) and microaggregates (53–250 μm) were enriched in C and N in CSW. Yield and macroaggregate-occluded C and N were negatively correlated. Yield and 5–25 cm free silt and clay C were positively correlated.

Conclusions

While organic matter in aggregate-occluded fractions is beneficial for soil C storage, it was correlated with lower grain yields, highlighting a potential tradeoff between yield and long-term C sequestration.

     

Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease, affecting the upper and/or lower motor neurons. However, extramotor symptoms can also occur; cognitive deficits are present in more than 40% of patients and 5–8% of ALS patients develop frontotemporal dementia. There is no effective treatment for ALS and median survival is 2–3 years after onset.Amyotrophic lateral sclerosis is a genetically heterogeneous disorder with monogenic forms as well as complex genetic etiology. Currently, complex genetic risk factors are of minor interest for routine diagnostic testing or counseling of patients and their families. By contrast, a monogenic cause can be identified in 70% of familial and 10% of sporadic ALS cases. The most frequent genetic cause is a noncoding hexanucleotide repeat expansion in the C9orf72 gene. In recent years, high-throughput sequencing technologies have helped to identify additional monogenic and complex risk factors of ALS.Genetic counseling should be offered to all ALS patients and their first- and possibly second-degree relatives, and should include information about the possibilities and limitations of genetic testing. Routine diagnostic testing should at least encompass the most frequently mutated disease genes (C9orf72, SOD1, TDP-43, FUS). Targeted sequencing approaches including further disease genes may be applied. Caution is warranted as the C9orf72 repeat expansion cannot be detected by routine sequencing technologies and testing by polymerase chain reaction (PCR) is failure-prone.Predictive testing is possible in families in which a genetic cause has been identified, but the limitations of genetic testing (i.?e., the problems of incomplete penetrance, variable expressivity and possible oligogenic inheritance) have to be explained to the families.     

Mutations in the gene encoding C8orf38 block complex I assembly by inhibiting production of the mitochondria-encoded subunit ND1

The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of “assembly factors” that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.