C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal Or Proteasomal Degradation in a Cell Type-Dependent Manner

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Article C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Stina Leskelä 1, Nadine Huber 1, Hannah Rostalski 1 , Teemu Natunen 2, Anne M. Remes 3,4 , Mari Takalo 2, Mikko Hiltunen 2 and Annakaisa Haapasalo 1,* 1 A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211 Kuopio, Finland; stina.leskela@uef.fi (S.L.); nadine.huber@uef.fi (N.H.); hannah.rostalski@uef.fi (H.R.) 2 Institute of Biomedicine, Yliopistonranta 1E, University of Eastern Finland, 70211 Kuopio, Finland; teemu.natunen@uef.fi (T.N.); mari.takalo@uef.fi (M.T.); mikko.hiltunen@uef.fi (M.H.) 3 Unit of Clinical Neuroscience, Neurology, University of Oulu, P.O. Box 8000, University of Oulu, 90014 Oulu, Finland; anne.remes@oulu.fi 4 MRC Oulu, Oulu University Hospital, P.O. Box 8000, University of Oulu, 90014 Oulu, Finland * Correspondence: annakaisa.haapasalo@uef.fi; Tel.: +358-40-355-2768; Fax: +358-17-163-025

Received: 16 August 2019; Accepted: 10 October 2019; Published: 10 October 2019

Abstract: Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.

Keywords: amyotrophic lateral sclerosis; autophagy; C9orf72; frontotemporal dementia; proteasomal degradation; ubiquitin-proteasome system

1. Introduction Frontotemporal dementia (FTD), the most common clinical phenotype of frontotemporal lobar degeneration (FTLD), is the third most common cause of early onset dementia in individuals under 65 years of age [1]. FTLD is clinically, neuropathologically, and genetically a heterogeneous group of neurodegenerative syndromes, and interestingly, it shares a partially overlapping genetic and molecular pathological background with amyotrophic lateral sclerosis (ALS) [2,3]. The most common genetic cause underlying both FTD and ALS is the GGGGCC-hexanucleotide repeat

Cells 2019, 8, 1233; doi:10.3390/cells8101233 www.mdpi.com/journal/cells Cells 2019, 8, 1233 2 of 18 expansion (HRE) in the C9orf72 gene [4–6]. The pathological mechanisms of the HRE underlying neurodegeneration are controversial, but are suggested to involve haploinsufficiency, leading to a decreased expression of the normal C9orf72 gene products (loss-of-function), as well as formation and accumulation of toxic RNA foci and dipeptide repeat (DPR) proteins that are directly generated from the expanded repeat (gain-of-toxic-function) [7–10]. Even though there is substantial evidence indicating that the main pathological mechanisms underlying HRE-associated FTD and ALS are related to gain-of-toxic-function, haploinsufficiency has also been suggested to contribute to the disease pathogenesis. Thus, neurodegeneration in HRE-linked FTD and ALS could involve co-operation between gain-of-toxic-function and loss-of-function mechanisms [11]. The normal physiological functions of the C9orf72 proteins, which may be influenced by the haploinsufficiency, are not yet well known. The C9orf72 gene produces three different transcript variants, which in humans are translated to two different protein isoforms, the long isoform A (~50 kDa) and the short isoform B (~25 kDa) [5]. Isoform B has been recently implicated in nucleo-cytoplasmic transport [12], while the isoform A contains a differentially expressed in normal and neoplastic cells (DENN) domain and thus is suggested to act as a guanosine exchange factor (GEF) for Rab-GTPases [13,14]. Accumulating experimental evidence indicates that the C9orf72 isoform A interacts with, and possibly activates, multiple different Rab-GTPases, such as Rab1, Rab3, Rab5, Rab7, Rab8, Rab10, Rab11, Rab13, Rab15, Rab29, and Rab39 [10,15–19], although the interaction seems to depend on the cell type as the expression of Rab-GTPases might display tissue specificity [20]. Thus, by regulating GDP/GTP exchange and subsequent activation of Rab-GTPases, the C9orf72 isoform A is suggested to regulate vesicular trafficking in the endosomal-lysosomal and autophagosomal-lysosomal pathways [13,15,21]. Autophagy and the ubiquitin-proteasome system (UPS) are essential pathways controlling proteostasis in cells, especially during stress conditions, such as those prevailing in diseased brain. These pathways are in charge of degrading unfolded, misfolded, or aggregated proteins. Neurons, as non-dividing cells with long axons and dendrites, are especially vulnerable to alterations in proteostasis [22]. In fact, defects in autophagy and UPS-mediated protein degradation pathways are suggested to contribute to the pathogenesis of many neurodegenerative diseases, including FTD and ALS [23]. In macroautophagy, hereafter referred to as autophagy, proteins are guided to degradation through autophagy receptor proteins, such as sequestosome 1 (p62/SQSTM1, hereafter p62). Autophagy can be induced by multiple environmental stimuli, such as nutrient deprivation, which initiates autophagic processes in order to provide a supply of metabolites for vital cellular functions, or by accumulation of misfolded or aggregated proteins [24,25]. Proteins are selected for degradation by ubiquitination and conjugated to an adaptor molecule, such as p62, which targets them to the double-membrane phagophore by binding to a membrane-bound receptor protein (e.g., LC3BII) on its inner surface. The expanding ends of the phagophore membrane ultimately fuse to create the autophagosome, which in the later phases of autophagy fuses with a lysosome to initiate degradation of its contents [26]. In the UPS, the proteins are guided to degradation also by ubiquitination to their lysine residues. The poly-ubiquitinated proteins are then targeted to the proteasome, where they are degraded to smaller peptides and amino acids, which can be further re-used in protein synthesis [23]. The UPS and autophagy-mediated protein degradation pathways are not mutually exclusive, but rather are interlinked and co-operate to maintain proteostasis in cells [23,27]. In C9orf72 HRE-associated FTD or ALS, the DPR proteins have been reported to hinder or block protein degradation through the UPS, emphasizing that the C9orf72 HRE causes toxicity by gain-of-toxic-function mechanisms [28,29]. On the other hand, C9orf72 loss-of-function due to haploinsufficiency has been suggested to lead to reduced autophagic degradation and subsequent accumulation of the DPR proteins, implicating that concomitant loss of C9orf72 function further exacerbates the effects of the gain-of-toxic-function mechanisms [30]. However, the potential role of the loss-of-function mechanisms in contributing to the toxicity of the C9orf72 HRE is still controversial. Recent studies have suggested that the C9orf72 protein isoform A regulates autophagy, but the reports Cells 2019, 8, 1233 3 of 18 have been partially controversial on whether reduced levels of C9orf72 lead to impaired or enhanced autophagy and at which steps on the autophagic pathway these effects take place [10,15–19,31–33]. In the present study, we have examined the role of C9orf72 in autophagy and UPS-mediated protein degradation pathways in neuronal cells and primary neurons. In addition, we have elucidated the yet-unknown regulation of the C9orf72 protein itself through autophagy and UPS-mediated protein degradation.

2. Materials and Methods

2.1. shRNA and cDNA Constructs To knockdown endogenous C9orf72, we used SMARTvector Lentiviral Mouse 3110043O21Rik shRNA containing mEF1a promoter and TurboRFP tag (Dharmacon, Lafayette, CO, USA, target sequence ATTATGTTAAGATCGCCCT, targets all transcripts). SMARTvector Non-targeting Control 1 with mEF1a promoter and TurboRFP (Dharmacon) was used as a control. To overexpress the C9orf72 protein isoform A, a cDNA construct encoding human C9orf72 isoform A containing a C-terminal green ﬂuorescent protein (GFP) tag (C9-A-GFP) was purchased from Origene (Rockville, MD, USA) and used as previously [34]. Empty plasmid (pcDNA3.1, Thermo Scientiﬁc, Carlsbad, CA, USA) was used as a control in the cDNA transfections.

2.2. Secondary Cell Culture, Transfection, and Treatments N2a mouse neuroblastoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Paisley, UK), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Lonza, Walkersville, MD, USA) at +37 ◦C and 5% CO2. A total of four µg of the plasmid DNA and 10 µL Lipofectamine 2000 reagent (Thermo Scientific, Carlsbad, CA, USA) were used per transfection of 400,000 cells according to the manufacturer’s instructions (scaled down proportionally for smaller cell amounts) to knockdown C9orf72 or to overexpress the C9orf72 protein isoform A. Fresh media were added 6 h post transfection when overexpressing the C9orf72 protein isoform A or after 24 h when using the shRNA to knockdown C9orf72. Cells were collected 48 (overexpression) or 72 h (knockdown) post transfection for analyses. To induce autophagy, serum starvation (ST) by culturing the cells in a medium without added FBS or treatment with 200 nM of rapamycin (Sigma, St. Louis, MO, USA) was used overnight. To assess autophagic flux, serum-starved cells were co-treated with 300 nM bafilomycin A1 (BafA1, Sigma, St. Louis, MO, USA) for the last 6 h to block the late phase of autophagy. In rapamycin-treated cells, the cells were co-treated with 200 nM rapamycin and 50 nM BafA1 overnight. To block protein degradation through the UPS, 10 µM lactacystin (Enzo Life Sciences, Farmingdale, NY, USA) was added overnight [15]. Dimethyl sulfoxide (DMSO, Hybri-Max, Sigma, St. Louis, MO, USA) was used as a vehicle control.

2.3. Immunofluorescence Studies In immunofluorescence experiments, glass coverslips were coated with 1:100 Matrigel GFR Basement Membrane Matrix (Corning, Bedford, MA, USA) for 1 h at room temperature (RT). N2a cells (ATCC) were simultaneously reverse co-transfected with either control or C9orf72 shRNA (3/4 of the total plasmid amount) and GFP-tagged LC3 construct (1/4 of the total plasmid amount). The GFP-LC3 plasmid was a kind gift from Prof. Kai Kaarniranta (UEF). Fresh media were changed 24 h after transfection and the cells were treated as described above. At 72 h post-transfection, the cells were fixed in 4% paraformaldehyde (PFA, Thermo Scientific, Rockford, IL, USA) for 10 min at RT and mounted with Vectashield Vibrance antifade mounting medium containing 40,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) to counterstain the nuclei. Images were taken with a LSM700 (ZEISS) confocal microscope and analyzed with ImageJ (Fiji, NIH). Quantification of the LC3-positive puncta was performed by first selecting shRNA-transfected cells (turboRFP-positive) for Cells 2019, 8, 1233 4 of 18 the analysis. After subtracting the background, the image was filtered by using the blur option, and correct threshold settings were chosen to ensure that the background signal was not detected as puncta but also that the real signal from the puncta was not lost. A particle analysis tool was used to quantify the number of puncta in each cell.

2.4. Primary Mouse Cortical Cell Culture, Virus Vector Transduction and Treatments Primary mouse cortical cell cultures were established as previously [35]. Brieﬂy, cortical cells were harvested from embryonic day 18 mouse embryos. A single-cell suspension was prepared from the dissected cortices by trypsin digestion and trituration. The cells were plated (400,000/well) on poly-d-lysine (PDL, Sigma, St. Louis, MO, USA)-coated 24-well plates in serum-free Neurobasal medium (Gibco, Grand Island, NY, USA) supplemented with 1 B27 (Gibco, Grand Island, NY, USA), × 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Half of the medium was changed every fourth day to feed the neurons. The cultures contain 90–95% of cortical neurons and 5–10% of astrocytes and other cells [36]. The neurons were matured for 11 days before the C9-A-GFP lentivirus-mediated transduction using 26 multiplicity of infection (MOI). The medium containing the viral particles was removed 24 h after transduction. The cells were cultured for 72 h after transduction before sample collection. To induce autophagy in the neuronal cultures, overnight treatment with 200 nM of rapamycin was used. To block the late phase of autophagy, BafA1 was used at a 50 nM concentration simultaneously with autophagy induction [37] to measure the autophagic ﬂux or separately for 6 h at 300 nM concentration to assess the basal autophagy. To block protein degradation through the UPS, treatment with 10 µM lactacystin was used overnight. DMSO was used as a vehicle control.

2.5. Protein Extraction from Cells and Western Blotting Proteins were extracted by scraping into T-PER, tissue protein extraction buffer, (Thermo Scientific, Rockford, IL, USA) supplemented with 1:100 protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA). The T-PER-soluble protein fractions were cleared by centrifugation of the cell lysates for 10 min at 10,000 g at +4 C. The resulting pellet was resuspended to 1% sodium dodecyl sulfate (SDS, × ◦ Sigma, St. Louis, MO, USA) extraction buffer (10 mM Tris-HCl (Sigma, St. Louis, MO, USA), 2 mM EDTA, (Sigma, St. Louis, MO, USA)) to collect the T-PER-insoluble protein fraction. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and 15–50 µg of proteins were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (NuPAGE Novex 4–12% Bis-Tris midi, Thermo Scientific, Carlsbad, CA, USA) for 1 h 45 min at 100 V. The proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Trans-Blot® Turbo™ Midi PVDF, Bio-Rad, Hercules, CA, USA) using Trans-Blot® Turbo™ Transfer System (Bio-Rad, 25 V, 30 min). After the transfer, unspecific binding sites on the membranes were blocked with 5% non-fat dry milk or bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) in 1 x Tris-buffered saline with 0.1% Tween 20 (Sigma, St. Louis, MO, USA) (TBST) for 1 h at RT. The protein bands were detected by incubating the membrane with protein-specific primary antibodies overnight at +4 ◦C and appropriate horse radish peroxidase-conjugated secondary (ECL™, anti-mouse IgG or anti-rabbit IgG, GE Healthcare, Buckinghamshire, UK, anti-mouse IgM, Thermo Scientific, Rockford, IL, USA) antibodies for 1 h at RT. The proteins were detected using enhanced chemiluminescence (ECL) detection reagents (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) and ChemiDoc™ XRS+ System (Bio-Rad). The intensities of the detected protein bands were quantified with Image Lab™ software (5.2.1, Bio-Rad) and normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin (loading controls). The membrane was stripped with a stripping buffer (Thermo Scientific, Rockford, IL, USA) for 10 min at RT, after which it was washed in 1 x TBST and reprobed with other antibodies. The following primary antibodies were used: anti-C9orf72 (1:500; Proteintech, Rosemont, IL, USA, 22637-1-AP); anti-SQSTM1/p62 (1:1000; Cell Signaling Technology (CST, Danvers, MA, USA 5114)); anti-LC3B (1:3000; Abcam, Cambridge, UK, ab51520); anti-poly-ubiquitinated proteins (FK1, 1:1000, Enzo Life Sciences, Farmingdale, NY, USA, Cells 2019, 8, 1233 5 of 18

BML-PW8805-0500); anti-LAMP2A (1:1000, Abcam, Cambridge, UK, ab18528); anti-beta-actin (1:1000, Abcam, Cambridge, UK, ab8226), and anti-GAPDH (1:5000, Abcam, Cambridge, UK, ab8245). The data are shown as % of the protein level compared to that in the control plasmid or control shRNA-transfected cells, or vehicle-treated cells (set to 100%).

2.6. Proteasomal Activity Measurement Proteasomal chymotrypsin-like activity was measured with a UBPBio kit (Aurora, CO, USA, J4110) or Abcam proteasomal activity kit (Cambridge, UK, ab107921) according to the kit instructions. Briefly, proteasomes from N2a cells transfected with control or C9orf72 shRNA or with pcDNA or C9-A-GFP constructs were extracted using a 0.5% NP-40 (Sigma, St. Louis, MO, USA) lysis buffer (prepared in distilled water) and centrifugation at 16,000 g for 20 min at +4 C. The protein concentrations were × ◦ measured using a Pierce BCA Protein Assay Kit. Ten µl of each proteasome lysate was incubated with the proteasomal substrate Succ-LLVY-AMC without or with the proteasomal inhibitor MG-132 (negative control) in two separate wells. The resulting fluorescence, indicating proteasomal activity, was measured at excitation/emission wavelength of 360 nm/460 nm, respectively, with an Infinite M200 (Tecan) plate reader. The specific activity of only the proteasomes, excluding the activity of other proteases present in the sample, was acquired by subtracting the fluorescence value of the corresponding MG-132-treated sample from the total fluorescence value in the sample without MG-132 treatment. The values were further normalized to the protein concentration of each sample and shown as % of control shRNA or pcDNA-transfected samples (set to 100%).

2.7. Statistical Analyses The data are shown as mean % standard error of mean (SEM) of control plasmid-, control ± shRNA-transfected or vehicle-treated cells as indicated. Statistical analyses were performed using GraphPad Prism5 (5.03). One-way analysis of variance (ANOVA) followed by Newman-Keuls post-hoc test or Mann-Whitney U test were used to test the statistical significance between sample groups. The level of statistical significance was defined as p 0.05. ≤ 3. Results Previous studies have suggested that the C9orf72 isoform A is a regulator of autophagy. However, whether knockdown of C9orf72 leads to enhanced or decreased autophagy is currently unclear and may depend on the cell type or on how the autophagy is modulated [10,15–19,31,32]. Furthermore, it is not completely clear if the site of action of the C9orf72 isoform A is in the early or later phases or both in the autophagic pathway [16,32,33]. Whether the C9orf72 proteins regulate the function of the UPS and whether they themselves are regulated by autophagy or UPS have not been studied before. Here, we have examined the effects of the knockdown or overexpression of C9orf72 protein isoform A on autophagy and UPS in N2a mouse neuroblastoma cells and mouse primary cortical neurons and investigated the regulation of C9orf72 protein isoform A itself via these protein degradation pathways.

3.1. C9orf72 Knockdown Leads to Decreased Autophagy Induction in N2a Cells During autophagy induction, phosphatidylethanolamine (PE) is conjugated with cytosolic LC3BI to form a membrane-bound lipidated LC3BII. Increased levels of LC3BII can therefore be used as a marker of autophagy induction and the number of autophagosomes present in the cells. Moreover, decreased levels of p62, a known autophagy receptor and substrate, reflect enhanced autophagic degradation [25,38]. Comparison of these factors can be used to help to distinguish between increased or decreased autophagy induction or autophagosomal clearance [25,38]. In N2a cells, 72-h shRNA treatment led to approximately a 40% decrease in the C9orf72 protein isoform A levels (Figure1A,B). Endogenous expression of the isoform B was not detectable in the N2a cells. Autophagy was induced by overnight serum starvation and BafA1 was used to block the fusion of autophagosomes with lysosomes and thus the later phases of the autophagosomal degradation pathway [25,38]. In the control shRNA-transfected N2a cells, the Cells 2019, 8, 1233 6 of 18

increased due to the conversion of LC3BI to LC3BII upon serum starvation (Figure 1C–E). Interestingly, in C9orf72 knockdown cells, the levels of LC3BI were significantly higher than those in the control shRNA-transfected cells upon autophagy induction (Figure 1C). In addition, the ratio of LC3BII/LC3BI did not significantly increase in C9orf72 knockdown cells similarly to control shRNA- transfected cells upon autophagy induction (Figure 1E). BafA1 treatment led to a similar strong Cells 2019, 8,accumulation 1233 of especially LC3BII and therefore increased LC3BII/LC3BI ratio in all the cells (Figure 6 of 18 1C–E). The p62 levels significantly decreased upon induction of autophagy in the control shRNA- LC3BII/LC3BItransfected ratio significantly cells, as expected. increased In the C9orf72 due to theknockdown conversion cells, ofhowever, LC3BI the toLC3BII decrease upon in p62 serum levels starvation was not evident upon serum starvation-induced autophagy, and the levels remained similar to the (Figure1C–E). Interestingly, in C9orf72 knockdown cells, the levels of LC3BI were significantly higher cells cultured in the normal medium. p62 levels similarly accumulated after the BafA1 treatment in than thosecontrol in the and control C9orf72 shRNA-transfected shRNA-transfected cells cells (Figure upon 1F). autophagy To determine induction whether (Figure knockdown1C). Inof addition, the ratio ofC9orf72 LC3BII has/LC3BI an effect did on not the significantly lysosomes, the increase immuno inblotC9orf72 was probedknockdown with an antibody cells similarly against to control shRNA-transfectedLAMP2A, a cells lysosomal upon protein. autophagy There induction were no significant (Figure1 changesE). BafA1 in LAMP2A treatment levels led in to C9orf72 a similar strong shRNA-transfected cells as compared to the control shRNA-transfected cells or between the different accumulation of especially LC3BII and therefore increased LC3BII/LC3BI ratio in all the cells (Figure1C–E). treatments (Figure 1A,G).

Figure 1. KnockdownFigure 1. Knockdown of C9orf72 of C9orf72 in N2a in N2a cells cells leads leads to to decreased decreased LC3BI LC3BI to LC3BII to LC3BII conversion conversion and p62 and p62 degradationdegradation during serumduring starvation-inducedserum starvation-induced autophagy. autophagy.( A)) A A representative representative Western Western blot of blot the of the total total protein lysates of N2a cells transfected with control (Ctrl) or C9orf72 (C9) shRNA showing the protein lysateslevels ofof N2athe endogenous cells transfected C9orf72 isoform with control A, LC3BI, (Ctrl) LC3BII, or C9orf72 p62, LAMP2A, (C9) shRNAand GAPDH showing (loading the levels of the endogenouscontrol) C9orf72 under normal isoform growth A, LC3BI, conditions, LC3BII, after p62,serum LAMP2A, starvation (ST) and or GAPDH ST and bafilomycin (loading control) A1 under normal growth conditions, after serum starvation (ST) or ST and bafilomycin A1 (BafA1) treatment. Overnight ST leads to autophagy induction as indicated by the conversion of LC3BI to LC3BII and the decrease in p62 levels in control shRNA-transfected cells but not in C9orf72 shRNA-transfected cells. The levels of p62 are restored and LC3BII robustly accumulates upon 300 nM BafA1 treatment for 6 h. Lysosomal LAMP2A levels did not show statistically significant alterations. Molecular weight markers are indicated on the left as kDa. An unspecific band, detected by the C9orf72 antibody and not affected by the shRNA, is indicated by an asterisk (*). Quantification of (B) the endogenous levels of C9orf72 isoform A, indicating an approximately 40% decrease in C9orf72 shRNA-transfected cells as compared to cells transfected with control shRNA, (C) LC3BI levels, (D) LC3BII levels, (E) ratio of LC3BII/LC3BI, (F) p62 levels, and (G) LAMP2A (unspecific band is indicated by *) levels. The protein levels were normalized to GAPDH levels in each sample. Data are shown as mean % SEM of the ± protein levels compared to those in the control cells. n = 9 from three independent experiments, except in p62 quantification, Ctrl shRNA Normal, n = 8 and LAMP2A quantifications, Ctrl shRNA ST n = 8; one-way ANOVA, Newman-Keuls, * p 0.05, ** p 0.01. ≤ ≤ Cells 2019, 8, 1233 7 of 18

(BafA1) treatment. Overnight ST leads to autophagy induction as indicated by the conversion of LC3BI to LC3BII and the decrease in p62 levels in control shRNA-transfected cells but not in C9orf72 Cells 2019shRNA-transfected, 8, 1233 cells. The levels of p62 are restored and LC3BII robustly accumulates upon 3007 of 18 nM BafA1 treatment for 6 h. Lysosomal LAMP2A levels did not show statistically significant alterations. Molecular weight markers are indicated on the left as kDa. An unspecific band, detected The p62 levels significantly decreased upon induction of autophagy in the control by the C9orf72 antibody and not affected by the shRNA, is indicated by an asterisk (*). Quantification shRNA-transfectedof B) the endogenous cells, as levels expected. of C9orf72 In the isoformC9orf72 A, indicatingknockdown an approximately cells, however, 40% the decrease decrease in in p62 levelsC9orf72 was shRNA-transfected not evident upon cells serum as compared starvation-induced to cells transfected autophagy, with control and the shRNA, levels C) remained LC3BI similarlevels, to the D cells) LC3BII cultured levels, in E the) ratio normal of LC3BII/LC3BI, medium. p62F) p62 levels levels, similarly and G) LAMP2A accumulated (unspecific after theband BafA1 is treatmentindicated in control by *) and levels.C9orf72 The proteinshRNA-transfected levels were norm cellsalized (Figure to GAPDH1F). To determinelevels in each whether sample. knockdown Data are of C9orf72shownhas as anmean eff ect% ± onSEM the of lysosomes,the protein levels the immunoblotcompared to those was in probed the control with cells. an antibodyn = 9 from againstthree LAMP2A,independent a lysosomal experiments, protein. except There in werep62 quantification, no significant Ctrl changes shRNA inNormal, LAMP2A n = 8 levels and LAMP2A in C9orf72 shRNA-transfectedquantifications, cells Ctrl as shRNA compared ST n = to 8; theone-way control ANOVA, shRNA-transfected Newman‒Keuls, cells * p or≤ 0.05, between ** p ≤ 0.01. the different treatments (Figure1A,G). ImmunofluorescentImmunofluorescent studies studies (Figure (Figure2A) 2A) revealed revealed significantly significantly fewer fewer GFP-LC3 GFP-LC3 puncta puncta after after serumserum starvation-induced starvation-induced autophagy autophagy in in the theC9orf72 C9orf72knockdown knockdown cells cells compared compared to to the the control control shRNA-transfectedshRNA-transfected cells cells (Figure (Figure2B), indicating 2B), indicati decreasedng decreased autophagosome autophagosome formation. BafA1formation. treatment BafA1 resultedtreatment in increased resulted numberin increased of the number LC3 puncta of the in bothLC3 controlpuncta andin bothC9orf72 controlshRNA-transfected and C9orf72 shRNA- cells. Thesetransfected findings, cells. in agreement These findings, with previous in agreement reports, support with theprevious idea that reports, knockdown support of C9orf72the idealeads that toknockdown a defective autophagy of C9orf72 inductionleads to a defective [16–18,31 ].autophagy induction [16–18,31].

FigureFigure 2. 2.Knockdown Knockdown of of C9orf72 C9orf72 in in N2a N2a cells cells leads leads to decreasedto decreased autophagosome autophagosome formation formation during during serumserum starvation-induced starvation-induced autophagy. autophagy. (A )A Representative) Representative fluorescent fluorescent microscope microscope images images of of the the control control oror C9orf72 C9orf72 shRNA shRNA and and GFP-LC3 GFP-LC3 plasmidplasmid co-transfectedco-transfected cells in normal normal growth growth medium, medium, upon upon ST- ST-inducedinduced autophagy autophagy or or combined combined treatment with STST andand BafA1.BafA1. ScaleScale bar:bar: 1010 µµm.m. (BB)) Quantification Quantification of ofthe LC3 punctapuncta withwith ImageJImageJsoftware software from from two two independent independent experiments. experiments. Four Four coverslips coverslips were were analyzedanalyzed/each/each treatment treatment group. group. Number Number of of the the analyzed analyzed cells cells in in total: total: Ctrl Ctrl shRNA shRNA Normal Normal 21, 21, C9 C9 shRNAshRNA Normal Normal 14, 14, Ctrl Ctrl shRNA shRNA ST ST 22, 22, C9 C9 shRNA shRNA ST ST 28, 28, Ctrl Ctrl shRNA shRNA ST ST+ BafA1+ BafA1 48, 48, C9 C9 shRNA shRNA ST ST+ + BafA1 22. Data are shown as mean SEM, one-way ANOVA, Newman-Keuls, * p 0.05, *** p 0.001. BafA1 22. Data are shown as mean± ± SEM, one-way ANOVA, Newman‒Keuls, ≤* p ≤ 0.05, ***≤ p ≤ 0.001. 3.2. Overexpression of C9orf72 Isoform A Does Not Affect Autophagy, But Its Levels are Decreased after Induction3.2. Overexpression of Autophagy of inC9orf72 N2a Cells Isoform A Does Not Affect Autophagy, but Its Levels are Decreased after Induction of Autophagy in N2a Cells Next, we assessed whether overexpression of the C9orf72 protein isoform A affects autophagy in N2aNext, cells. Aswe theassessed commercially whether availableoverexpression antibodies of th typicallye C9orf72 produce protein aisofor highm degree A affects of unspecific autophagy binding,in N2a wecells. transfected As the commercially N2a cells with available a cDNA antibodi constructes typically encoding produce C9-A-GFP, a high todegree allow of a unspecific reliable examinationbinding, we of transfected the levels and N2a eff ectscells ofwith the C9orf72a cDNA isoformconstruct A, encoding or an empty C9-A-GFP, vector (pcDNA). to allow The a reliable cells wereexamination serum-starved of theovernight levels and and effects treated of the with C9orf72 BafA1 isoform for 6 h A, before or an sample empty collectionvector (pcDNA). as before. The No cells significantwere serum-starved differences inovernight the levels and of treated LC3BI, with LCBII, BafA1 ratio for of LC3BII6 h before/LC3BI, sample or thecollection levels ofas p62 before. were No detected in N2a cells overexpressing the C9orf72 isoform A (Figure3A,C,F) as compared to control cells, suggesting that overexpression of C9orf72 protein isoform A does not influence autophagy. Moreover, Cells 2019, 8, 1233 8 of 18 significant differences in the levels of LC3BI, LCBII, ratio of LC3BII/LC3BI, or the levels of p62 were detected in N2a cells overexpressing the C9orf72 isoform A (Figure 3A and C–F) as compared to control cells, suggesting that overexpression of C9orf72 protein isoform A does not influence autophagy. Moreover, in co-immunoprecipitation experiments, we did not detect interaction of the C9orf72 protein isoform A with LC3B or p62 under normal conditions or upon autophagy induction by serum starvation (data not shown). Interestingly, a statistically significant decrease in the levels of the C9orf72 protein isoform A (Figure 3A,B) was detected after serum starvation-induced autophagy in N2a cells, suggesting that it might undergo autophagy-mediated degradation. However, when we further tested this hypothesis by blocking the autophagosomal degradation by BafA1, we did not detect increased C9orf72 protein isoform A levels (Figure 3A,B) in N2a cells in a similar manner to those of p62, a known autophagy substrate (Figure 3F). The C9orf72 isoform A levels were also significantly decreased when autophagy was induced by treatment of the N2a cells with rapamycin, which specifically targets the mammalian target of rapamycin complex 1 (mTORC1) (Figure 4A,B). Again, the levels of the C9orf72 isoform A were not restored after BafA1 treatment in rapamycin-treated cells, as would be expected if the proteins were regulated through autophagic degradation (Figures 4A,B). Together, the results from these experiments suggest that the decreased C9orf72 protein isoform A levels upon autophagy induction are not caused by their autophagy-mediated degradation in N2a cells. Cells 2019, 8, 1233 8 of 18 In contrast to the N2a cells, however, a statistically significant increase in the levels of C9orf72 isoform A after rapamycin and BafA1 treatment in primary neurons was detected (Figures 4C,D), suggestingin co-immunoprecipitation that in these cells, experiments, the C9orf72 we didprotein not detect isoform interaction A may ofundergo the C9orf72 autophagy-mediated protein isoform A degradationwith LC3B or upon p62 underrapamycin-induced normal conditions autophagy. or upon These autophagy results inductionimply differential by serum regulation starvation of (data the C9orf72not shown). protein isoform A levels in different cell types upon modulation of autophagy.

Figure 3. N2a cells overexpressing the C9orf72 isoform A do not show alterations in autophagy but Figuredisplay 3. decreased N2a cells C9orf72overexpressing isoform the A levels C9orf72 after isoform serum A starvation. do not show (A) Aalterations representative in autophagy Western but blot displayof the total decreased protein C9orf72 lysates ofisoform N2a cells A levels transfected after serum with thestarvation. control pcDNAA) A representative or C9orf72 isoform Western A-GFP blot (C9-A-GFP) plasmids showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and GAPDH (loading control) under normal growth conditions, after serum starvation (ST) or ST and BafA1 treatment. ST overnight leads to similarly increased LC3BII and decreased p62 levels in both the control and the C9-A-GFP overexpressing cells. The levels of p62 and LC3BII accumulate in a similar manner after 300 nM BafA1 treatment for 6 h in the pcDNA and C9-A-GFP-transfected cells. C9-A-GFP levels are decreased after ST treatment and are not restored after BafA1 treatment. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). Quantification of (B) C9-A-GFP, (C) LC3BI, (D) LC3BII, (E) LC3BII/LC3BI ratio, and (F) p62 levels. Data are shown as mean % SEM of the protein levels compared to those in the control cells ± from three independent experiments. n = 8, except C9-A-GFP ST + BafA1, n = 5. One-way ANOVA, Newman-Keuls, * p 0.05, *** p 0.001. ≤ ≤ Interestingly, a statistically significant decrease in the levels of the C9orf72 protein isoform A (Figure3A,B) was detected after serum starvation-induced autophagy in N2a cells, suggesting that it might undergo autophagy-mediated degradation. However, when we further tested this hypothesis by blocking the autophagosomal degradation by BafA1, we did not detect increased C9orf72 protein isoform A levels (Figure3A,B) in N2a cells in a similar manner to those of p62, a known autophagy substrate (Figure3F). The C9orf72 isoform A levels were also significantly decreased when autophagy was induced by treatment of the N2a cells with rapamycin, which specifically targets the mammalian target of rapamycin complex 1 (mTORC1) (Figure4A,B). Again, the levels of the C9orf72 isoform Cells 2019, 8, 1233 9 of 18

of the total protein lysates of N2a cells transfected with the control pcDNA or C9orf72 isoform A-GFP (C9-A-GFP) plasmids showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and GAPDH (loading control) under normal growth conditions, after serum starvation (ST) or ST and BafA1 treatment. ST overnight leads to similarly increased LC3BII and decreased p62 levels in both the control and the C9- A-GFP overexpressing cells. The levels of p62 and LC3BII accumulate in a similar manner after 300 Cells 2019nM, 8BafA1, 1233 treatment for 6 h in the pcDNA and C9-A-GFP-transfected cells. C9-A-GFP levels are9 of 18 decreased after ST treatment and are not restored after BafA1 treatment. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by A werean asterisk not restored (*). Quantification after BafA1 of treatment B) C9-A-GFP, in rapamycin-treated C) LC3BI, D) LC3BII, cells, E) LC3BII/LC3BI as would be ratio, expected and F if) the proteinsp62 werelevels. regulated Data are throughshown as autophagicmean % ± SEM degradation of the protein (Figure levels4A,B). compared Together, to those the results in the control from these experimentscells from suggest three independent that the decreased experiments. C9orf72 n = protein 8, except isoform C9-A-GFP A levels ST + uponBafA1, autophagy n = 5. One-way induction are notANOVA, caused Newman by their‒ autophagy-mediatedKeuls, * p ≤ 0.05, *** p ≤ 0.001. degradation in N2a cells.

Figure 4. Induction of autophagy by rapamycin leads to decreased C9orf72 protein isoform A levels, Figure 4. Induction of autophagy by rapamycin leads to decreased C9orf72 protein isoform A levels, which are not restored in N2a cells but show a significant increase in mouse cortical neurons upon BafA1 which are not restored in N2a cells but show a significant increase in mouse cortical neurons upon treatment. (A) A representative Western blot of the total protein lysates extracted from transfected N2a BafA1 treatment. (A) A representative Western blot of the total protein lysates extracted from cells showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading control). Treatment with transfected N2a cells showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading 200 nM rapamycin 50 nM Bafilomycin A1 (BafA1) overnight leads to decreased levels of C9-A-GFP control). Treatment± with 200 nM rapamycin ± 50 nM Bafilomycin A1 (BafA1) overnight leads to and the levels are not restored after BafA1 treatment. Molecular weight markers are indicated on decreased levels of C9-A-GFP and the levels are not restored after BafA1 treatment. Molecular weight the left as kDa. An unspecific band, detected by the C9orf72 antibody, is indicated by an asterisk markers are indicated on the left as kDa. An unspecific band, detected by the C9orf72 antibody, is (*). (B) Quantification of the C9-A-GFP protein levels in N2a cells normalized to those of β-actin. indicated by an asterisk (*). (B) Quantification of the C9-A-GFP protein levels in N2a cells normalized Data are shown as mean % SEM of the protein levels compared to those in the vehicle-treated cells ± fromto those two of independent β-actin. Data experiments. are shown asn mean= 6, except% ± SEM C9-A-GFP of the protein rapamycin, levels ncompared= 5, one-way to those ANOVA, in the Newman-Keuls,vehicle-treated cells * p from0.05. two (C) independent A representative experiments. Western blotn = 6, of except total protein C9-A-GFP lysates rapamycin, extracted n from = 5, ≤ lentivirus-transducedone-way ANOVA, Newman primary‒Keuls, cortical * neuronsp ≤ 0.05. showing(C) A representative the levels of theWestern C9-A-GFP, blot of LC3BI, total LC3BII,protein p62,lysates and extractedβ-actin (loadingfrom lentivirus-transduced control). Molecular primar weighty cortical markers neurons are indicated showing on the the levels left as of kDa. the AnC9- unspecificA-GFP, LC3BI, band LC detected3BII, p62, by and the C9orf72β-actin (loading antibody control). is indicated Molecular by an weight asterisk markers (*). (D) are Quantifications indicated on ofthe C9-A-GFP left as kDa. protein An unspecific levels normalized band detected to those by oftheβ -actin.C9orf72 Data antibody are shown is indica as meanted by % an SEMasterisk of the(*). ± protein(D) Quantifications levels compared of C9-A-GFP to those in protein the vehicle levels treated normalized cells from to those three independentof β-actin. Data experiments. are shownn as= 11, one-way ANOVA, Newman-Keuls, * p 0.05, ** p 0.01. ≤ ≤ In contrast to the N2a cells, however, a statistically significant increase in the levels of C9orf72 isoform A after rapamycin and BafA1 treatment in primary neurons was detected (Figure4C,D), suggesting that in these cells, the C9orf72 protein isoform A may undergo autophagy-mediated Cells 2019, 8, 1233 10 of 18 degradation upon rapamycin-induced autophagy. These results imply differential regulation of the Cells 2019, 8, 1233 10 of 18 C9orf72 protein isoform A levels in different cell types upon modulation of autophagy. mean % ± SEM of the protein levels compared to those in the vehicle treated cells from three 3.3. C9orf72 Proteinindependent Isoform experiments. A Levels n = Are11, one-way Increased ANOVA, after Newman Proteasomal‒Keuls, * Inhibition p ≤ 0.05, ** p in≤ 0.01. N2a Cells and Primary Neurons 3.3. C9orf72 Protein Isoform A Levels Are Increased after Proteasomal Inhibition in N2a Cells and Primary As weNeurons observed that the C9orf72 isoform A protein levels were decreased during autophagy, but not restored afterAs we blockage observed ofthat autophagosomal the C9orf72 isoform degradation A protein levels by were BafA1 decreased in N2a during cells, autophagy, we asked whether they couldbut be not regulated restored after through blockage the of UPS. autophagosomal To this end, degradation N2a cells by transfected BafA1 in N2a with cells, the we C9-A-GFP asked were treated overnightwhether they with could the proteasomalbe regulated through inhibitor the UPS. lactacystin. To this end, A significantN2a cells transfected increase with in the C9orf72 C9-A- isoform A levels wasGFP observed were treated (Figure overnight5A,B). In with addition, the proteasomal C9orf72 proteininhibitor isoformlactacystin. A accumulatedA significant increase in the SDS-solublein C9orf72 isoform A levels was observed (Figure 5A,B). In addition, C9orf72 protein isoform A fraction, containingaccumulated large in amountsthe SDS-soluble of accumulating fraction, cont poly-ubiquitinatedaining large amounts proteins, of accumulating upon lactacystin poly- treatment (Figure5A).ubiquitinated Isoform A proteins, strongly upon accumulated lactacystin intreatment a similar (Figure manner 5A). Isoform in both A T-PER- strongly and accumulated SDS-soluble in a fractions and also insimilar cultured manner mouse in both primary T-PER- and cortical SDS-soluble neurons fractions (Figure and5 alsoC,D). in cultured Taken together, mouse primary these cortical results suggest that C9orf72neurons proteins (Figure are 5C,D). regulated Taken bytogether, the UPS these in result boths suggest N2a cells that andC9orf72 mouse proteins primary are regulated cortical by neurons. the UPS in both N2a cells and mouse primary cortical neurons.

Figure 5. C9orf72 protein isoform A levels are elevated after proteasomal inhibition in N2a cells Figure 5. C9orf72 protein isoform A levels are elevated after proteasomal inhibition in N2a cells and and mousemouse primary primary cortical cortical neurons. neurons. (A (A) )A A representative representative Western Western blot of blot the total of the protein total lysates protein lysates extractedextracted in T-PER in andT-PER SDS and extractionSDS extraction bu buffersffers fromfrom transfected transfected N2a N2a cells cellsor (C) ortransduced (C) transduced mouse mouse primary corticalprimary neuronscortical neurons showing showing the the levels levels of of thethe C9-A-GFP, C9-A-GFP, poly-ubiquitinat poly-ubiquitinateded (Poly-Ub.) (Poly-Ub.) proteins, proteins, GAPDH or β-actin (loading control). Treatment with 10 µM lactacystin overnight leads to GAPDH or β-actin (loading control). Treatment with 10 µM lactacystin overnight leads to significantly significantly increased levels of C9-A-GFP proteins and accumulation of poly-ubiquitinated proteins, increasedespecially levels of to C9-A-GFP the SDS-soluble proteins fraction and in accumulationboth N2a cells and of poly-ubiquitinatedcortical neurons. Molecular proteins, weight especially to the SDS-solublemarkers are fractionindicated inon boththe left N2a as kDa. cells An and unsp corticalecific band neurons. detected by Molecular the C9orf72 weightantibody markersis are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (B,D) Quantification of C9-A-GFP protein levels in the T-PER fraction of N2a cells or primary mouse cortical neurons normalized to those of GAPDH or β-actin. Data are shown as mean % SEM of the protein levels compared to those in the vehicle-treated cells from two independent ± experiments. n = 8 for all other samples, except n = 6 for C9-A-GFP-transduced neurons treated with vehicle, Mann-Whitney U test, * p 0.05, *** p 0.001. ≤ ≤ Cells 2019, 8, 1233 11 of 18

indicated by an asterisk (*). (B) and (D) Quantification of C9-A-GFP protein levels in the T-PER fraction of N2a cells or primary mouse cortical neurons normalized to those of GAPDH or β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle-treated cells from two independent experiments. n = 8 for all other samples, except n = 6 for C9-A-GFP-transduced neurons treated with vehicle, Mann‒Whitney U test, * p ≤ 0.05, *** p ≤ 0.001. Cells 2019, 8, 1233 11 of 18 3.4. Decreased C9orf72 Protein Isoform A Levels in Serum-Starved N2a Cells Are Restored by Proteasomal Inhibition3.4. Decreased C9orf72 Protein Isoform A Levels in Serum-Starved N2a Cells Are Restored by ProteasomalWe next Inhibition tested the hypothesis whether the decrease in the C9orf72 protein isoform A levels duringWe serum next tested starvation-induced the hypothesis whetherautophagy the decreasein N2a cells in the could C9orf72 be proteinexplained isoform by its A levelstargeting during to proteasomalserum starvation-induced degradation. autophagyInduction of in N2aautophagy cells could by serum be explained starvation by its in targeting N2a cells to again proteasomal led to significantlydegradation. decreased Induction C9orf72 of autophagy isoform by A serum protein starvation levels (Figure in N2a 6A,B). cells again When led the to proteasomal signiﬁcantly degradationdecreased C9orf72 was blocked isoform in Athese protein serum-starved levels (Figure cell6sA,B). using When lactacystin, the proteasomal the C9orf72 degradation protein isoform was Ablocked levels were in these restored serum-starved back to the cells basal using levels lactacystin, (Figure 6A the and C9orf72 B), suggesting protein that isoform the decrease A levels in were the C9orf72restored protein back to isoform the basal A levelslevels (Figureduring 6autophagyA,B), suggesting is due thatto their the proteasomal decrease in thedegradation C9orf72 protein in N2a cells.isoform A levels during autophagy is due to their proteasomal degradation in N2a cells.

Figure 6. Decreased C9orf72 protein isoform A levels upon serum starvation-induced autophagy are Figurerestored 6. byDecreased lactacystin C9orf72 treatment protein in N2aisoform cells. A (levelsA) A representativeupon serum starvation-induced Western blot of the autophagy total protein are restoredlysates extracted by lactacystin in T-PER treatment and SDS in extraction N2a cells. bu (Aff)ers A representative from transfected Western N2a cells blot showing of the total the levelsprotein of lysatesC9-A-GFP, extracted LC3BI, in LC3BII, T-PER p62,and poly-ubiquitinatedSDS extraction buffe proteinsrs from (poly-Ub.),transfected and N2a GAPDH cells showing (loading the control). levels ofOvernight C9-A-GFP, serum LC3BI, starvation LC3BII, (ST) p62, leads poly-ubiquitinated to decreased C9-A-GFP proteins protein (poly-Ub.), levels and that GAPDH are restored (loading upon control).10 µM lactacystin Overnight treatment. serum starvation The levels (ST) of p62leads also to slightlydecreased increase, C9-A-GFP but LC3BI protein or IIlevels levels that do notare restoredshow major upon changes 10 µM after lactacystin lactacystin trea treatmenttment. The in levels serum-starved of p62 also cells. slightly Molecular increase, weight but markersLC3BI or are II levelsindicated do not on show the left major as kDa. changes An unspecific after lactacystin band detected treatment by in the serum-starved C9orf72 antibody cells. Molecular is indicated weight by an markersasterisk (*).are (indicatedB) Quantification on the left of theas kDa. C9-A-GFP An unsp proteinecific levels band normalizeddetected by tothe those C9orf72 of GAPDH antibody from is indicatedtwo independent by an asterisk experiments. (*). (B) Quantification Data are shown of asthe mean C9-A-GFP % SEM prot ofein the levels protein normalized levels compared to those of to ± GAPDHthose in thefrom vehicle two independent treated cells. experiments.n = 6 for all samples,Data are one-wayshown as ANOVA, mean % Newman-Keuls,± SEM of the protein ** p levels0.01, ≤ compared*** p 0.001. to those in the vehicle treated cells. n = 6 for all samples, one-way ANOVA, Newman‒ ≤ 3.5. ProteasomalKeuls, ** p ≤ Activity0.01, *** p Is ≤Slightly 0.001. Decreased in N2a Cells Upon C9orf72 Knockdown but Not Changed After C9orf72 Protein Isoform A Overexpression 3.5. Proteasomal Activity Is Slightly Decreased in N2a Cells Upon C9orf72 Knockdown but Not Changed After PreviousC9orf72 Protein studies Isoform by others A Overexpression have shown that C9orf72 interacts with ubiquilin 1 and 2 proteins, which are linked to protein degradation through the UPS [15]. Therefore, we next assessed the effects of the Previous studies by others have shown that C9orf72 interacts with ubiquilin 1 and 2 proteins, C9orf72 protein isoform A on proteasomal function. Here, we used both shRNA-mediated knockdown which are linked to protein degradation through the UPS [15]. Therefore, we next assessed the effects of endogenous C9orf72 or overexpression of the C9orf72 protein isoform A in N2a cells and measured of the C9orf72 protein isoform A on proteasomal function. Here, we used both shRNA-mediated the proteasomal activity with the help of the fluorescent proteasomal substrate Succ-LLVY-AMC. Each knockdown of endogenous C9orf72 or overexpression of the C9orf72 protein isoform A in N2a cells extracted proteasome lysate from the transfected cells was incubated with the proteasomal substrate without or with the proteasomal inhibitor MG-132 (negative control) in two separate wells per each experiment. The specific activity of only the proteasomes, excluding the activity of other proteases present in the sample, was acquired by subtracting the fluorescence value of the corresponding MG-132-treated lysate from the total fluorescence value in the lysate without the MG-132 treatment. Proteasomal activity was slightly, but statistically significantly, decreased in C9orf72 knockdown cells, Cells 2019, 8, 1233 12 of 18 and measured the proteasomal activity with the help of the fluorescent proteasomal substrate Succ- LLVY-AMC. Each extracted proteasome lysate from the transfected cells was incubated with the proteasomal substrate without or with the proteasomal inhibitor MG-132 (negative control) in two separate wells per each experiment. The specific activity of only the proteasomes, excluding the activity of other proteases present in the sample, was acquired by subtracting the fluorescence value of the corresponding MG-132-treated lysate from the total fluorescence value in the lysate without the MG-132 treatment. Proteasomal activity was slightly, but statistically significantly, decreased in Cells 2019, 8, 1233 12 of 18 C9orf72 knockdown cells, whereas there was no significant difference in the proteasomal activity in cells overexpressing the C9orf72 isoform A compared to control cells (Figure 7A,B). As whereasoverexpression there was of C9orf72 no significant isoform di ffAerence did not in thehave proteasomal an effect on activity the proteasomal in cells overexpressing activity and thethe C9orf72C9orf72 isoformknockdown A compared effect was to controlmodest, cells these (Figure data7A,B). imply As that overexpression C9orf72 may of C9orf72not have isoform a major A didregulatory not have role an einff ectUPS on function, the proteasomal at least in activity N2a cells. and the C9orf72 knockdown effect was modest, these data imply that C9orf72 may not have a major regulatory role in UPS function, at least in N2a cells.

Figure 7. C9orf72 knockdown mildly decreases whereas overexpression of the C9orf72 isoform A doesFigure not 7. a ffC9orf72ect the proteasomalknockdown activitymildly indecreases N2a cells. whereas Quantification overexpression of proteasomal of the C9orf72 activity (Aisoform) in cells A transfecteddoes not affect with the the proteasomal control (Ctrl) activity or the C9orf72in N2a cells. (C9) Quantification shRNA. Data are of proteasomal shown as mean activity % SEM (A) in of cells the ± activitytransfected in control with the shRNA control samples. (Ctrl) or n =the6 fromC9orf72 two (C9) independent shRNA. Data experiments, are shown Mann-Whitney as mean % ± SEM U test, of *thep activity0.05. (B in) in control cells overexpressing shRNA samples. the n C9-A-GFP. = 6 from two Data independent are shown as experiments, mean % SEM Mann of‒ theWhitney activity U ≤ ± intest, pcDNA * p ≤ 0.05. samples. (B) inn cells= 6 fromoverexpressing two independent the C9-A-GFP. experiments, Data are one-way shown ANOVA, as mean Newman-Keuls,% ± SEM of the notactivity significant. in pcDNA samples. n = 6 from two independent experiments, one-way ANOVA, Newman‒ Keuls, not significant. 3.6. C9orf72 Protein Isoform A Levels Are Unchanged After BafA1 Treatment in N2a Cells, but Decreased in Mouse Primary Cortical Neurons 3.6. C9orf72 Protein Isoform A Levels Are Unchanged After BafA1 Treatment in N2a Cells, but Decreased in MouseFinally, Primary we Cortical investigated Neurons whether the levels of the C9orf72 isoform A are regulated by autophagy in basal conditions without the induction of autophagy in N2a cells or primary cortical neurons. N2a Finally, we investigated whether the levels of the C9orf72 isoform A are regulated by autophagy cells overexpressing the C9orf72 isoform A were treated with BafA1 for 6 h. While the BafA1 treatment in basal conditions without the induction of autophagy in N2a cells or primary cortical neurons. N2a expectedly resulted in the accumulation of p62 and LC3BII, no significant changes were observed in cells overexpressing the C9orf72 isoform A were treated with BafA1 for 6 h. While the BafA1 the levels of C9orf72 protein isoform A in these conditions in N2a cells (Figure8A,B). In contrast, in treatment expectedly resulted in the accumulation of p62 and LC3BII, no significant changes were the mouse primary cortical neurons, the BafA1 treatment led to a significant decrease in the C9orf72 observed in the levels of C9orf72 protein isoform A in these conditions in N2a cells (Figure 8A,B). In isoform A levels, while the p62 levels increased in a similar manner to those in N2a cells (Figure8C,D). contrast, in the mouse primary cortical neurons, the BafA1 treatment led to a significant decrease in In neurons, BafA1 treatment did not majorly affect LC3BII levels. These data suggest that there might the C9orf72 isoform A levels, while the p62 levels increased in a similar manner to those in N2a cells be cell type-specific differences in the basal autophagy and the regulation of C9orf72 levels. (Figure 8C,D). In neurons, BafA1 treatment did not majorly affect LC3BII levels. These data suggest that there might be cell type-specific differences in the basal autophagy and the regulation of C9orf72 levels.

Cells 2019, 8, 1233 13 of 18 Cells 2019, 8, 1233 13 of 18

Figure 8. Blockade of the basal autophagy with BafA1 does not signiﬁcantly change the C9orf72 protein

isoform A levels in N2a cells but leads to its decreased levels in mouse primary cortical neurons. (A)A representativeFigure 8. Blockade Western of blot the of basal the total autophagy protein lysateswith BafA1 from transfecteddoes not significantly N2a cells showing change the the levels C9orf72 of C9-A-GFP,protein isoform LC3BI, LC3BII,A levels p62, in andN2a βcells-actin but (loading leads to control). its decreased Treatment levels with in 300 mouse nM Bafilomycin primary cortical A1 (BafA1)neurons. for 6(A h) does A representative not alter C9-A-GFP Western protein blot levelsof the but tota increasesl protein p62 lysates levels, from as expected. transfected Molecular N2a cells weightshowing markers the levels are indicated of C9-A-G onFP, the leftLC3BI, as kDa. LC3B AnII, unspecificp62, and β band-actin detected (loading by control). the C9orf72 Treatment antibody with is indicated300 nM Bafilomycin by an asterisk A1 (BafA1) (*). (B) Quantificationfor 6 h does not ofalter the C9-A-GFP C9-A-GFP protein protein levels levels but normalized increases p62 to those levels, of asβ-actin. expected. Data Molecular are shown weight as mean marker % sSEM are indicated of the protein on the levels left as compared kDa. An unspecific to those in band the vehicle detected ± treatedby the cells C9orf72 from threeantibody independent is indicated experiments. by an asteriskn = 9, (*). except (B) Quantification in vehicle n = 8, of Mann-Whitney the C9-A-GFP U protein test, notlevels significant. normalized (C) A to representative those of β-actin. Western Data blotare shown of the total as mean protein % ± lysates SEM of from the protein lentivirus-transduced levels compared mouseto those primary in the corticalvehicle treated neurons cells showing from three the levels independent of C9-A-GFP, experiments. LC3BI, n LC3BII, = 9, except p62, in and vehicleβ-actin n = 8, (loadingMann‒ control).Whitney U Treatment test, not significant. with 300 nM (C) BafA1 A representative for 6 h leads Western to significantly blot of the decreased total protein levels lysates of C9-A-GFPfrom lentivirus-transduced proteins, while p62 levelsmouse are primary increased, cortical as expected. neurons Molecular showing the weight levels markers of C9-A-GFP, are indicated LC3BI, onLC3BII, the left p62, as kDa. and An β-actin unspecific (loading band control). detected Treatment by the C9orf72 with 300 antibody nM BafA1 is indicated for 6 h leads by an toasterisk significantly (*). (Ddecreased) Quantification levels of theC9-A-GFP C9-A-GFP proteins, protein while levels p62 normalized levels are increased, to those of asβ -actin.expected. Data Molecular are shown weight as meanmarkers % SEM are ofindicated the protein on levelsthe left compared as kDa. toAn those unsp inecific thevehicle band detected treated cells by fromthe C9orf72 two independent antibody is ± experiments.indicated byn an= 8, asterisk except (*). C9-A-GFP (D) Quantification BafA1 n = 7, of Mann-Whitney the C9-A-GFP prot U test,ein *** levelsp 0.001.normalized to those of ≤ β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle treated 4. Discussioncells from two independent experiments. n = 8, except C9-A-GFP BafA1 n = 7, Mann‒Whitney U test, In*** the p present≤ 0.001. study, we have examined the role of the C9orf72 protein isoform A in the autophagy and UPS-mediated protein degradation pathways and how it itself is regulated via these pathways in neuronal4. Discussion cells. The C9orf72 proteins mainly reside in the cytosol and they may also be secreted [15]. Additionally,In the thepresent C9orf72 study, isoform we have A has examined been shown the role to localize of the inC9orf72 the lysosomes protein isoform [10,15,31 A,33 in,39 the]. Previousautophagy studies and have UPS-mediated indicated that protein the C9orf72 degradation isoform pathways A forms a and protein how complex it itself withis regulated Smith-Magenis via these pathways in neuronal cells. The C9orf72 proteins mainly reside in the cytosol and they may also be secreted [15]. Additionally, the C9orf72 isoform A has been shown to localize in the lysosomes

Cells 2019, 8, 1233 14 of 18 syndrome chromosome region candidate 8 (SMCR8) and WD repeat-containing protein 41 (WDR41) to regulate autophagy induction [16,18,31,33]. SMCR8, but not WDR41, protein levels are co-regulated with the levels of C9orf72 in cells [32] and a near-stoichiometric presence of these proteins has been suggested [18]. Moreover, the C9orf72 proteins have been suggested to interact with many proteins of the autophagy initiation complex, such as Unc-51 like autophagy activating kinase (ULK1), autophagy-related protein 13 (ATG13), and RB1-inducible coiled-coil protein 1 (RB1CC1/FIP200) [40], further suggesting a regulatory role in autophagy. The C9orf72 proteins have also been shown to interact with proteins like ubiquilin 1 and 2, which are linked to UPS functions [15]. The data on whether modulation of the C9orf72 levels leads to enhanced or impaired autophagy has been controversial and appear to depend on the model system used and how the autophagy is modulated. In some studies, reduction of the C9orf72 protein levels was shown to lead to impaired basal autophagy, whereas in other studies autophagy defects are detected only after induction of autophagy with for example torin 1 or rapamycin. Moreover, the phase of the autophagic pathway at which the C9orf72 proteins display their effects appears to vary in different studies [15–18,31,40]. Webster et al. [17] have reported increased autophagic activity in non-neuronal HeLa and HEK293 cells after C9orf72 overexpression. We did not detect alterations in autophagy upon overexpression of the C9orf72 isoform A in neuronal N2a cells. In contrast, C9orf72 knockdown in N2a cells led to impaired autophagy, which is in accordance with several previous studies [16–18,31]. The C9orf72 isoform A has been suggested to represent the predominantly expressed isoform in mouse central nervous system (CNS) [10]. In line with this, we found that N2a cells do not express detectable levels of the isoform B. Therefore, we hypothesize that reduced levels of the C9orf72 isoform A underlie the observed defect in autophagy. The impairment in autophagy induction was suggested by the findings that knockdown of C9orf72 led to impaired LC3BI to LC3BII conversion and degradation of p62, markers that can be used to assess autophagy initiation, in serum-starved N2a cells [38]. In addition, significantly fewer GFP-LC3-containing autophagosomes were detected in C9orf72 knockdown N2a cells with serum starvation-induced autophagy when compared to control cells. Our data showed that the effect of the C9orf72 knockdown on LC3BII/LC3BI ratio, as detected by Western blotting, was milder than that on the number of GFP-LC3-positive autophagosomal vesicles observed by immunofluorescence microscopy. It is possible that these differences might reflect a possibly deleterious effect of C9orf72 knockdown on the trafficking of the autophagosomal vesicles, while the effect on autophagosome biogenesis might be more modest. Alternatively, these differences could be due to differential analysis of a total cell population in the Western blots vs. analysis of individual transfected cells in the immunofluorescence microscopy. Furthermore, we did not observe any changes in lysosomal marker protein LAMP2A levels, implying that C9orf72 knockdown may not majorly influence the late phases in the autophagosomal-lysosomal pathway in N2a cells. On the other hand, the finding that LAMP2A protein levels remained unchanged does not rule out possible morphological changes in the lysosomes or changes in the lysosome number in cells, which could be revealed by immunofluorescence studies. For example, Shi et al. [41] found no changes in LAMP2A levels in the lysosomal membrane fraction from C9orf72 repeat expansion-carrying FTD patient-derived induced motor neurons, but immunofluorescence and electron microscopy studies revealed fewer lysosomes, which contained a higher concentration of LAMP2A proteins on their membrane surface. Altogether, these results corroborate the recent reports showing similarly impaired autophagy induction upon C9orf72 deficiency in other cell types. Furthermore, our findings together with the previous studies support the idea that the haploinsufficiency, taking place in the FTD and ALS patients carrying the C9orf72 HRE and resulting in the decreased C9orf72 levels, may contribute to defective autophagy and accumulation of, e.g., aggregated TDP-43 and DPR proteins, which represent the typical pathological hallmark changes in the CNS of the C9orf72 HRE carriers. Our data further suggest that the C9orf72 proteins mainly regulate protein degradation via the autophagosomal pathway and do not play a major role in Cells 2019, 8, 1233 15 of 18 controlling the UPS in neuronal cells, as overexpression of the C9orf72 protein isoform A did not alter and the C9orf72 knockdown only mildly decreased proteasomal activity in N2a cells. An interesting novel finding was that in N2a cells, serum starvation- or rapamycin-induced autophagy led to significantly decreased levels of the C9orf72 isoform A. These levels were not restored after blocking the autophagosomal degradation by BafA1 treatment similarly to those of p62, a known autophagy substrate. Furthermore, blocking the basal autophagic degradation using BafA1 did not significantly change C9orf72 protein levels in N2a cells, while p62 levels increased as expected. Together, these data suggest that the C9orf72 proteins do not undergo autophagy-mediated degradation in N2a cells. The C9orf72 protein levels were previously shown to increase after BafA1 and the proteasomal inhibitor MG-132 treatment in HEK293 cells, implying that in these non-neuronal cells, C9orf72 levels could be regulated through both autophagy and UPS [32]. We also observed that blocking the proteasomal protein degradation using lactacystin resulted in the increased levels of the C9orf72 protein isoform A in N2a cells and primary cortical neurons. Moreover, the decreased C9orf72 protein isoform A levels upon serum starvation-induced autophagy in N2a cells were restored by treatment with lactacystin, suggesting that the C9orf72 protein isoform A could be guided to UPS-mediated degradation during autophagy. Given the suggested role of C9orf72 isoform A in promoting autophagy induction, these results may suggest a feedback loop between autophagy and the UPS to regulate the C9orf72 isoform A levels during autophagy and to maintain the balance in cellular proteostasis. In mouse cortical neurons, in contrast to N2a cells, co-treatment with rapamycin and BafA1 led to significantly elevated levels of the C9orf72 isoform A. Moreover, while BafA1 treatment alone (without autophagy induction) did not influence the C9orf72 isoform A levels in N2a cells, it significantly decreased the C9orf72 isoform A levels in primary cortical neurons. The reason for this observation during basal autophagy in neurons currently remains unclear but may support the notion that there is a crosstalk between the autophagic and UPS-mediated degradation pathways and that upon blockade of autophagy, C9orf72 proteins may alternatively be targeted to proteasomal degradation. Collectively, our results suggest that the C9orf72 isoform A levels may be differentially regulated via UPS and/or autophagy depending on the cell type during basal conditions and upon different autophagic stimuli. Taken together, the present data confirm the previously suggested role for the C9orf72 protein isoform A in the regulation of autophagy and underscore the key role for C9orf72 in controlling autophagy induction in neuronal cells. Moreover, to our knowledge, we show for the first time that the C9orf72 protein isoform A itself can undergo regulation through both autophagy and proteasomal degradation pathways and suggest that this regulation may differ depending on the cell type and prevailing conditions. However, these findings warrant further confirmation in other models, such as cells with stable C9orf72 knockdown or expression.

Author Contributions: Conceptualization, S.L., M.H., and A.H.; methodology, S.L., N.H., H.R., T.N., and M.T.; investigation, S.L. and M.T.; resources, A.M.R., M.H., and A.H.; data curation, S.L., M.H., and A.H.; writing—original draft preparation, S.L. and A.H.; writing—review and editing, S.L., N.H., H.R., T.N., A.M.R., M.T., M.H., and A.H.; visualization, S.L. and A.H.; supervision, A.M.R., M.T., M.H., and A.H.; project administration, A.H.; funding acquisition, S.L., A.M.R., M.H., and A.H. Funding: This work was supported by the Academy of Finland, under grant numbers 315459, 315460, 307866, and 288659; Yrjö Jahnsson Foundation under grant number 20187070; ALS tutkimuksen tuki ry. registered association; Sigrid Jusélius Foundation; VTR grant 5772816 of Kuopio University Hospital; the Strategic Neuroscience Funding of the University of Eastern Finland; Neurocenter Finland—AlzTrans pilot project. This publication is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 740264. Acknowledgments: We thank Kai Kaarniranta (Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio) for generously providing the GFP-LC3 plasmid. We also wish to thank Janne Capra, PhD, (Cell and Tissue Imaging Unit, Unit of Biomedicine, University of Eastern Finland, Kuopio) for technical assistance and advice on the immunofluorescent analyses of the LC3 puncta. Petra Mäkinen (Unit of Biomedicine, University of Eastern Finland, Kuopio) and Erasmus exchange students Julia List and Mireia Cartró Font are acknowledged for their assistance with N2a cell cultures and treatments. S.L., N.H., and H.R. are PhD Cells 2019, 8, 1233 16 of 18 students in the Doctoral Programs of Molecular Medicine (DPMM) and GenomMed (H.R.) of the University of Eastern Finland. Conflicts of Interest: The authors have no conflict of interest to report.

Abbreviations

ALS Amyotrophic lateral sclerosis BafA1 Baﬁlomycin A1 CNS Central nervous system C9FTD/ALS Concomitant FTD/ALS caused by C9orf72 hexanucleotide repeat expansion C9orf 72 Chromosome 9 open reading frame 72 gene C9orf72 C9orf72 protein coded by C9orf72 DENN Diﬀerentially expressed in normal and neoplastic cells DPR Dipeptide repeat FTD Frontotemporal dementia FTLD Frontotemporal lobar degeneration GEF Guanosine exchange factor HRE Hexanucleotide repeat expansion LC3B Microtubule-associated proteins 1A/1B light chain 3B mTOR Mammalian target of rapamycin p62/SQSTMI Sequestosome 1 PE Phosphatidylethanolamine SMCR8 Smith-Magenis syndrome chromosome region candidate 8 TDP-43 TAR DNA-binding protein 43 UPS Ubiquitin-proteasome system WDR41 WD repeat-containing protein 41

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