Abstract
The most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion (HRE) in C9ORF72 that contributes to neurodegeneration by both loss-of-function (decreased C9ORF72 protein levels) and gain-of-function (e.g. dipeptide repeat protein production) mechanisms. Although therapeutics targeting the gain-of-function mechanisms are in clinical development, it is unclear if these will be efficacious given the contribution of C9ORF72 loss-of-function processes to neurodegeneration. Moreover, there is a lack of therapeutic strategies for C9ORF72 ALS/FTD with demonstrated efficacy in vivo. Here, we show that small molecule inhibition of PIKFYVE kinase rescues both loss- and gain-of-function C9ORF72 disease mechanisms in vivo. We find that the reduction of C9ORF72 in mouse motor neurons leads to a decrease in early endosomes. In contrast, treatment with the PIKFYVE inhibitor apilimod increases the number of endosomes and lysosomes. We show that reduced C9ORF72 levels increases glutamate receptor levels in hippocampal neurons in mice, and that apilimod treatment rescues this excitotoxicity-related phenotype in vivo. Finally, we show that apilimod also alleviates the gain-of-function pathology induced by the C9ORF72 HRE by decreasing levels of dipeptide repeat proteins derived from both sense and antisense C9ORF72 transcripts in hippocampal neurons in vivo. Our data demonstrate the neuroprotective effect of PIKFYVE kinase inhibition in both gain- and loss-of-function murine models of C9ORF72 ALS/FTD.
Main text
The C9ORF72 repeat expansion causes neurodegeneration through loss- and gain-of-function mechanisms [1–3]. Reduced C9ORF72 levels alter endosomal trafficking [3, 4], autophagy [5, 6], and lysosomes in vitro [3, 7–9], and increase glutamate receptors on neurons [3]. C9ORF72 ALS/FTD (C9-ALS/FTD) gain-of-function mechanisms include neurotoxic dipeptide repeat proteins (DPRs) generated by repeat-associated non-AUG translation [3, 10–15].
Although antisense oligonucleotide therapeutics targeting sense C9ORF72 transcripts are undergoing clinical testing, their efficacy may be limited given the contribution of C9ORF72 loss-of-function processes to neurodegeneration [3, 5, 16] and the fact that both sense and antisense C9ORF72 transcripts generate DPRs. Problematically, there is a lack of other targets whose perturbation can rescue C9-ALS/FTD disease processes in vivo. We recently performed a phenotypic screen to identify small molecules that rescue the survival of C9-ALS/FTD induced motor neurons (iMNs) [3]. PIKFYVE kinase inhibitors such as apilimod potently rescued C9-ALS/FTD iMN survival [3]. Suppression of PIKFYVE using antisense oligonucleotides rescued C9-ALS iMN survival, confirming PIKFYVE as the therapeutic target [3].
PIKFYVE is a Class-III Phosphatidylinositol-5-kinase (PI5K) that synthesizes PI(3,5)P2 from PI3P [17]. PIKFYVE inhibition promotes endosomal maturation by increasing PI3P levels, and PI3P is also critical for autophagosome formation and engulfment of proteins designated for degradation [18, 19]. Therefore, PIKFYVE regulates cellular processes that are disrupted in C9-ALS/FTD, suggesting that altering PIKFYVE activity could modulate C9-ALS/FTD processes in patients. Here, we examine (1) the effects of the C9ORF72 HRE and (2) the ability of PIKFYVE inhibition to rescue gain- and loss-of-function of C9ORF72 ALS/FTD disease processes in vivo.
To determine if C9ORF72 deficiency alters endosomal trafficking in vivo, we used mice with approximately 50% reduced C9ORF72 levels in heterozygotes and a complete loss of C9ORF72 in homozygotes [20, 21]. We and others have shown that even modest changes in the number of intracellular vesicles such as endosomes, lysosomes, or autophagosomes by less than 50% can impair protein trafficking and degradation in neurons and reduce neuronal survival in neurodegenerative disease models [3, 22–25]. Moreover, mutations in one copy of GBA, which encodes a lysosomal enzyme, increase the risk of developing Parkinson’s disease by 20-30-fold [26]. Therefore, to increase the sensitivity of our analyses, we quantified endosome and lysosome numbers on a per cell basis in similar fashion to previous studies [3, 24, 25]. However, we included quantification of the median values and variation on a per animal basis to assess reliability of our results. We found that hippocampal neurons of C9orf72+/- mice, which mimic the ~50% reduction in C9ORF72 levels in C9-ALS/FTD patients, had fewer EEA1+ early endosomes than controls (Fig. 1A-C). Immunoblotting on total brain samples showed that PIKFYVE levels were similar between control and C9orf72-/- mice (Fig. 1D, E), indicating that differences in PIKFYVE levels did not cause the reduction in EEA1+ vesicles.
It remains unclear whether the endosome and lysosome changes caused by C9ORF72 deficiency drive neurodegeneration or are adaptive compensatory changes that prevent neurodegeneration [3]. Because PIKFYVE inhibition promotes C9-ALS/FTD iMN survival [3], determining its effects on endosomes and lysosomes could elucidate this issue. Injection of apilimod directly to the hippocampus of C9orf72+/- mice increased the number of EEA1+ vesicles in both neurons and GFAP+ astrocytes (Fig. 1F-K).
C9ORF72-deficient human and mouse motor neurons have fewer LAMP1+ vesicles (lysosomes) than controls [3]. Apilimod increased LAMP1+ vesicle number in hippocampal neurons in C9orf72/- mice 24 hours after treatment (Fig. 1L-N). Thus, in hippocampal neurons in adult mice, PIKFYVE inhibition and C9ORF72 deficiency have opposing effects on endosomal and lysosomal numbers, suggesting that having fewer endosomes and/or lysosomes due to reduced C9ORF72 levels may drive neurodegeneration and increasing endosomes and/or lysosomes by PIKFYVE inhibition may rescue neurodegeneration.
C9ORF72-deficient mouse and human spinal motor neurons have increased glutamate receptor levels [3]. Because the C9ORF72 HRE can induce neurodegeneration in the hippocampus, cortex, and spinal cord [27–30], we have used stereotactic injections into the hippocampus to measure the effects of C9ORF72 deficiency and PIKFYVE inhibition on NMDA-induced excitotoxicity [3]. Direct injection of NMDA into the hippocampus causes significantly greater excitotoxic injury in C9ORF72-deficient mice than controls [3].
To determine if the exacerbated excitotoxic injury in C9ORF72-deficient mice might result from increased glutamate receptor levels in brain regions affected by the C9orf72 HRE, we measured NR1 (NMDA receptor subunit) and GLUR6/7 (kainate receptor subunit) levels in the hippocampus and frontal cortex of C9orf72+/+ and C9orf72+/- mice. Changes in glutamate receptor levels by less than 50% [3, 31–33] and as little as 5% [34] can have a strong effect on neuronal function, sensitivity to neurotransmitters, and animal survival. Therefore, to increase the sensitivity of our analyses, we quantified glutamate receptor levels on a per cell basis in similar fashion to previous studies [3, 32, 33]. However, we included quantification of the median values and variation on a per animal basis to assess reliability of our results.
Immunohistochemistry showed that NR1 and GLUR6/7 levels were increased in the hippocampus and frontal cortex of C9orf72+/- mice compared to C9orf72+/+ mice (Fig. 2A-F, J-O). To confirm that reduced C9ORF72 levels lead to increased glutamate receptor levels in vivo, we harvested post-synaptic densities from the brains of C9orf72-/- mice, which have undetectable C9ORF72 levels [21]. Post-synaptic density preparations were enriched for PSD-95 and not enriched for presynaptic proteins including Synaptophysin and intracellular proteins including p53 (Fig. 2G). Immunoblotting confirmed that NR1 levels were increased in post-synaptic densities of C9orf72-/- mice, indicating they may affect neurotransmission (Fig. 2H, I). Thus, reduced C9ORF72 levels lead to increased glutamate receptor levels in multiple C9ORF72 HRE-affected brain regions in vivo.
Apilimod rescues the increased susceptibility to NMDA-induced excitotoxicity in the hippocampus of C9orf72+/- mice [3]. However, it is unclear if this is accomplished in part through direct reversal of C9ORF72 loss-of-function disease processes, for example by lowering glutamate receptor levels, or through some unrelated mechanism that prevents cell death. In C9orf72+/- mice, apilimod treatment lowered NR1 and GLUR6/7 levels in the CA1 region and dentate gyrus (Fig. 2P-U). Thus, PIKFYVE inhibition can lower glutamate receptor levels in C9ORF72-deficient tissue. Consistent with the notion that apilimod-modulated NR1 levels would affect the NMDA-sensitivity of hippocampal neurons, apilimod dose-dependently reduced NMDA-induced neurodegeneration in the hippocampus after 48 hours (Fig. 2V). Thus, apilimod can rescue increased glutamate receptor levels, a C9ORF72 loss-of-function disease phenotype that increases susceptibility to glutamate-induced excitotoxicity.
C9-BAC mice harboring a human C9ORF72 gene containing 100-1000 GGGGCC repeats recapitulate C9-ALS/FTD gain-of-function processes by producing DPRs that aggregate in neurons [35]. We previously showed that a single injection of apilimod decreased the number of poly-glycine-arginine (poly(GR))+ punctae in the dentate gyrus of C9-BAC mice [3]. However, poly(GR)+ DPRs are generated from the sense C9ORF72 transcript, and we did not determine if PIKFYVE inhibition can reduce aggregates containing poly-proline-arginine (poly(PR)), which is made from the antisense C9ORF72 transcript [36]. Additionally, we did not assess the effect of PIKFYVE inhibition on poly-glycine-proline (poly(GP))+ DPR aggregates, which are more prevalent than arginine-containing DPRs in C9-ALS/FTD patients and highly neurotoxic in mice [37]. Unlike the nuclear-localized poly(GR)+ and poly(PR)+ aggregates, poly(GP)+ aggregates are predominantly cytosolic [36], making it unclear if mechanisms that reduce poly(GR)+ aggregate levels would lower poly(GP)+ aggregates. Since multiple DPR species can cause neurotoxicity [38], a key measure of therapeutic efficacy against C9-ALS/FTD gain-of-function processes is the ability to diminish multiple DPRs.
Changes in DPR levels by less than 50% can have a strong effect on neuronal survival [3] and animal behavior [28]. Therefore, to increase the sensitivity of our analyses, we quantified DPR punctae levels on a per cell basis in similar fashion to previous studies [3, 15]. However, we included quantification of the median values and variation on a per animal basis to assess reliability of our results.
Apilimod increased the number of EEA1+ vesicles in C9-BAC mice after 48 hours (Fig. 3A-C), indicating that PIKFYVE inhibition results in similar endosomal changes in the presence or absence of the C9ORF72 repeat expansion. 48 hours after injection into the hippocampus, apilimod reduced the number of poly(GP)+ (Fig. 3D-F) and poly(PR)+ (Fig. 3G-I) punctae in the dentate gyrus. Thus, increases in endosome numbers correlate with reduced DPR levels, and PIKFYVE inhibition can reduce nuclear and cytoplasmic DPR aggregates derived from both sense and antisense C9ORF72 transcripts in vivo.
Surprisingly, C9-BAC mice contained slightly elevated hippocampal NR1 and GLUR6/7 levels (Fig. 3J-O), indicating that gain- and loss-of-function C9ORF72 processes can both lead to increased glutamate receptor levels. Thus, C9-ALS/FTD gain- and loss-of-function processes can have similar effects on glutamate receptor levels in vivo, reinforcing the importance of curtailing both gain- and loss-of-function processes.
Here, we show that C9ORF72 insufficiency leads to fewer endosomes and increased surface-bound glutamate receptor levels in mice, which could sensitize neurons to glutamate-induced excitotoxicity. PIKFYVE inhibition reverses endosome, lysosome, and glutamate receptor changes induced by reduced C9ORF72 function in mice. It also significantly reduces both cytosolic and nuclear, and both sense and antisense transcript-derived DPR aggregates in hippocampal neurons. Thus, our data provide some of the first evidence that a pharmacological intervention, PIKFYVE inhibition, can rescue both gain- and loss-of-function C9ORF72 disease processes in vivo.
Methods
Animal Care
All animal use and care were in accordance with local institution guidelines of the University Medical Center Utrecht and approved by the Dierexperimenten Ethische Commissie Utrecht with the protocol number DEC 2013.I.09.069. Wild-type C57BL6/J (strain: 000664), C9ORF72 KO (C57BL/6J-3110043021Rikem5Lutzy/J, strain: 027068), and C9-BAC (C57BL/6J-Tg(C9orf72_i3)112Lutzy/J, strain: 023099) were purchased from Jackson Laboratories. Mice were housed in standard conditions with food and water ad libitum in the conventional vivarium at the University of Southern California. All animal use and care were in accordance with local institution guidelines of the University of Southern California and the IACUC board of the University of Southern California with the protocol numbers 20546 and 11938.
Direct injection of apilimod
Mice were anesthetized with i.p. ketamine (100 mg/kg) and xylazine (10 mg/kg), and body temperature kept at 36.9 ± 0.1 °C with a thermostatic heating pad. Mice were placed in a stereotactic apparatus (ASI Instruments, USA) and the head is fixed accordingly. A burr hole was drilled, and an injection needle (33 gauge) was lowered into the hippocampus between CA1 and the dentate gyrus (AP −2.0, ML +1.5, DV −1.8). Apilimod (0.3 μl of 0.5, 3, or 20 μM in phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments). The needle was left in place for an additional 8 min after the injection. Animals were euthanized 24h-48h later (as stated in the text).
Immunohistochemistry
C9orf72+/-, C9-BAC, and wildtype controls were transcardially perfused with phosphate buffered saline (PBS) and subsequently with 4% formaldehyde. Cryoprotection occurred in 20% sucrose. After snap freezing, tissue was sectioned by cryostat at 20 μm thickness and stained with the following primary antibodies: NR1 (MAB363, EMD Millipore), GLUR6/7 (04-921, EMD Millipore), EEA1 (sc-6415, N19, G4, Santa Cruz), LAMP1 (ab24170, Abcam), NeuN (MAB374, EMD Millipore), ChAT (AB144P, Millipore Sigma), poly(PR) (23979-1-AP, ProteinTech), and poly(GP) (24494-1-AP, ProteinTech). Antigen retrieval by sodium citrate occurred before NR1 staining, and with Target Retrieval Solution (pH 9, Agilent, Dako) for the poly(GP) staining. Images were collected using a Zeiss LSM780 or LSM800 confocal microscope. Glutamate subunit intensity measurements occurred with ImageJ where the mean cytosolic intensity was subtracted by a background measurement collected near to the measured neuron, and then divided by the background measurement. The scientist performing the glutamate receptor subunit intensity, EEA1 vesicle, LAMP1 vesicle, poly(PR)+ punctae, and poly(GP)+ punctae quantification was blinded to the genotype or treatment condition of the samples.
Sections used for staining and quantification from all experiments were a random selection from across the hippocampus. For images of EEA1+ and LAMP1+ vesicles the images were adjusted for publication to ensure that the background intensity was similar. Confocal microscopy images of poly(GP)+ or poly(PR)+ punctae were adjusted for brightness and contrast for optimal visualization of the poly(GP)+ or poly(PR)+ punctae; this occurred to the same extent for the vehicle- and apilimod-treated side of the hippocampus. No corrections were applied to the images of glutamate receptor stainings, as these are based on intensity of signal measurements.
Western Blot
Mouse spinal cord tissue was collected in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Membrane samples were obtained by the Plasma Membrane Isolation Kit (Abcam) according to the manufacturer’s instructions. Postsynaptic density extraction occurred by a protocol published previously [39]. In brief, tissues were homogenized in cold Sucrose Buffer (320 mM Sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, 30 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, and protease inhibitor cocktail (Roche)) using a tissue grinder and then spun down at 500 g for 6 min at 4 °C. The supernatant was re-centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was collected as the ‘Total’ fraction, and the pellet was resuspended in cold Triton buffer (50 mM HEPES pH 7.4, 2 mM EDTA, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail (Roche)) and then spun down at 30,000 RPM using a Beckman rotor MLA-130 for 40 min at 4 °C. The supernantant was collected as the ‘Triton’ fraction and the pellet was resuspended in DOC buffer (50 mM HEPES pH 9.0, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 20 μM ZnCl2, 1% sodium deoxycholate and protease inhibitor cocktail (Roche)) and collected as the “DOC”, PSD-enriched fraction. Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at 4 °C. The gel was transferred onto PVDF membrane (GE Healthcare) using Trans-Blot Semi-Dry transfer cell (Biorad). The membrane was blocked with 5% milk in 0.1% PBS-Tween 20 (PBS-T) (Sigma-Aldrich), incubated with primary antibodies overnight at 4 °C, washed three times with 0.1% PBS-T, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz). After three washes with 0.1% PBS-T, blots were visualized using an Amersham ECL Western Blotting Detection Kit (GE) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and developed on X-ray film (Genesee Scientific). The following primary antibodies were used: goat anti-Actin-HRP (Santa Cruz, cat. no. sc-47778 HRP, 1:4,000), mouse anti-NR1 (Novus, cat. no. NB300118, 1:2,000), mouse anti-PSD-95 (Thermo, cat. no. MA1-045, 1:1,000), mouse anti-p53 (Cell Signaling, cat. no. 2524S, 1:1,000), mouse anti-Synaptophysin (Sigma, cat. no. S5768, 1:1000), anti-mouse HRP (Cell Signaling, cat. no. 7076S, 1:5,000), and anti-rabbit HRP (Cell Signaling, cat. no. 7074S, 1:5,000).
Statistics
Analysis was performed with the statistical software package Prism Origin v.7.0a and v8.0a (GraphPad Software). The normal distribution of datasets was tested by the D’Agostino-Pearson omnibus normality test. Differences between two groups were analyzed using a two-tailed Student’s t test, unless the data was non-normally distributed for which two-sided Mann-Whitney testing was used. Differences between more than two groups were analyzed by one way-ANOVA with Tukey correction for multiple testing unless the data was non-normally distributed for which Kruskal-Wallis testing was used. Mean and standard deviation or standard error of the mean were used for normally distributed datasets, and the median and interquartile range were used for not normally distributed datasets. Significance was assumed at p < 0.05. Details of the statistical analyses for each panel are included in the figure legends.
Declarations
Ethics approval
All animal use and care were in accordance with local institution guidelines of the University of Southern California and the IACUC board of the University of Southern California with the protocol numbers 20546 and 11938.
Consent for publication
Not applicable.
Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests
W-HC is an employee of AcuraStem.
JKI is a co-founder of AcuraStem. JKI declares that he is bound by confidentiality agreements that prevent him from disclosing details of his financial interests in this work.
Funding
This work was made possible by NIH grants R00NS077435, R01NS097850, 1R44NS097094-01A1, and 1R44NS105156-01, US Department of Defense grant W81XWH-15-1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Alzheimer’s Drug Discovery Foundation, the Association for Frontotemporal Degeneration, the Harrington Discovery Institute, the Tau Consortium, the Pape Adams Foundation, the Frick Foundation for ALS Research, the John Douglas French Alzheimer’s Foundation, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to JKI. JKI is a New York Stem Cell Foundation-Robertson Investigator and a Richard N.Merkin Scholar. CS and DK are funded by the Undergraduate Research Associates Program (University of Southern California). RJP was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12-08, 15-05), and VICI grant Netherlands Organisation for Scientific Research (NWO). PC was supported by NIH grant MH 1135457. YW and BZ are supported by the NS090904, the NS100459, and the Foundation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease reference no. 16 CVD 05. KAS is funded by a development award from the Muscular Dystrophy Association.
Authors’ contributions
KAS designed and planned the experiments, performed the initial experiments, supervised experiments, analyzed the data, and wrote the manuscript. YL identified the PIKFYVE target. CS, AS, NK, DK, KAG, and YL performed the experiments, and quantified readouts. YW performed the direct injection experiments, and MC performed drug treatment surgeries (unpublished results) that enabled the study. SL and WHC performed post-synaptic density isolations and performed all Western blot experiments. RJP, KS and VRV provided tissue, and supervised the work. PC and BZ designed experiments, and supervised the work. YS and YL provided unpublished results that enabled the study. JKI conceived the study, designed and supervised the experiments, and wrote the manuscript. All authors agreed with the final version of the manuscript.
Acknowledgements
We thank the lab members of the Ichida lab for their useful feedback, and the University of Southern California Department of Animal Resources staff for their outstanding care.
Footnotes
Staats, K. A. Kim.Staats.Work{at}gmail.com, Seah C. cseah{at}usc.edu, Sahimi, A. asahimi{at}usc.edu, Wang, Y. yaomingw{at}usc.edu, Koutsodendris, N. nkoutsodendris{at}gmail.com, Lin, S. shaoyuli{at}usc.edu, Kim, D. david.k.2015{at}gmail.com, Chang, W-H. wchang{at}acurastem.com, Gray, K. A. kag262{at}georgetown.edu, Shi, Y. shiyingxiao{at}gmail.com, Li, Y. yichen.li{at}st-annes.ox.ac.uk, Chateau, M. mchateau{at}usc.edu, Vangoor, V. R. V.Vangoor{at}umcutrecht.nl, Senthilkumar, K. K.Senthilkumar-2{at}umcutrecht.nl, Pasterkamp, R. J. R.J.Pasterkamp{at}umcutrecht.nl, Cannon, P. PCannon{at}usc.edu, Zlokovic, B.V. Zlokovic{at}usc.edu, Ichida, J. K. Ichida{at}usc.edu
List of abbreviations
- ALS
- amyotrophic lateral sclerosis
- CA
- Cornu Ammonis
- C9-ALS/FTD
- C9orf72 ALS/FTD
- C9-BAC mice
- C9orf72 containing 100-1000 GGGGCC repeats mice
- DG
- dentate gyrus
- DPR
- dipeptide repeat protein
- EEA1
- Early Endosome Antigen 1
- FTD
- frontotemporal dementia
- GP
- glycine-proline repeat DPR
- GR
- glycine-arginine repeat DPR
- HRE
- hexanucleotide repeat expansion
- HRP
- horseradish peroxidase
- iPSC
- induced pluripotent stem cell
- iMN
- induced motor neuron
- LAMP1
- Lysosomal Associated Membrane Protein 1
- NMDA
- N-methyl-D-aspartate
- PBS
- phosphate buffered saline
- PI5K
- Phosphatidylinositol 5-kinase
- PR
- proline-arginine repeat DPR
- RAN
- repeat-associated non-AUG