Ever since repeat expansions in the C9ORF72 gene were identified as a major cause of amyotrophic lateral sclerosis and frontotemporal dementia, researchers have been debating whether the disease is caused by loss of the normal protein or accumulation of toxic aggregates. Most previous data has trended toward the latter. However, a new study in the February 5 Nature Medicine makes the case that both mechanisms contribute, with a drop in levels of C9ORF72 protein leaving neurons more susceptible to the toxic effects of the repeat expansion.
Researchers led by Justin Ichida at the University of Southern California in Los Angeles used pluripotent stem cells to show that induced motor neurons with little C9ORF72 died more readily than control cells when exposed to stressors such as nutrient depletion or excess glutamate. Adding back a normal C9ORF72 gene rescued survival. In addition, low C9ORF72 levels hampered endosomal trafficking, leading to a dearth of lysosomes. That in turn allowed toxic dipeptide repeat proteins (DPRs) made from the expanded RNA to pile up. Inhibiting an endosomal kinase, PIKFYVE, restored lysosomal maturation, reduced excitotoxicity, and lowered the number of aggregates. “PIKFYVE inhibition appears to modulate several ALS disease processes, and may have therapeutic potential,” Ichida told Alzforum.
Other researchers were enthusiastic. “This is a landmark study,” Jiou Wang at Johns Hopkins University in Baltimore wrote to Alzforum. He praised the thoroughness of the experiments and controls, noting that they represent a heroic effort to address multiple aspects of C9ORF72-associated toxicity. Robert Baloh at Cedars-Sinai Regenerative Medicine Institute, Los Angeles, agreed. “[This] adds further evidence that the disease cannot be simply looked at from the perspective of one potential toxic mechanism, such as RNA granules or dipeptide repeats, but rather should be looked at as resulting from a combination of gain- and loss-of-function effects within and across cell types in the nervous system,” he wrote.
Gain-of-function mechanisms have garnered the most attention from researchers, with numerous studies linking C9ORF72 expanded RNA foci and DPRs to toxicity in cell cultures (Dec 2014 news; Jul 2017 news; Feb 2018 news). However, C9ORF72 expansions have little effect in many mouse models, suggesting other factors may contribute to the disease, as well (May 2016 news; see also June 2017 news). Meanwhile, the repeat expansion squelches production of C9ORF72 protein, raising the possibility that loss of normal protein function could factor into the disease. Researchers have paid less attention to this idea, because C9ORF72 knockout mice do not lose motor neurons or have movement problems (Jun 2015 news).
Stress Unmasks C9ORF72 Haploinsufficiency
Ichida and colleagues decided to take a closer look at C9ORF72’s role. Joint first authors Yingxiao Shi and Shaoyu Lin confirmed that induced motor neurons (iMNs) generated from ALS and FTD patients carrying the expansion made about half as much C9 protein as did iMNs from controls. Under normal culture conditions, these C9 patient iMNs survived as well as control iMNs, the authors found. Under stress from nutrient withdrawal or excess glutamate, however, C9 cells died off more quickly.
Was lack of C9ORF72 protein to blame for this vulnerability? To test this, the authors upped C9ORF72 expression in patient and control iMNs by expressing the normal gene off a retrovirus. This rescued survival in patient iMNs and had no effect on controls. Then, the researchers knocked out one or both copies of C9ORF72 in control iMNs. These cells died as quickly as C9 patient iMNs upon exposure to excess glutamate. In addition, transcriptional profiles from C9-deficient iMNs, C9 patient iMNs, and postmortem tissue from C9 patients all resembled each other, but varied from the profile of healthy controls.
Gain and Loss of Function Both Converge on Excitotoxicity
Ichida and colleagues next tried to dissect how low C9ORF72 levels caused problems. Because of the glutamate sensitivity of patient iMNs, they measured NMDA and AMPA glutamate receptors on the cell surface. They found that C9 patient iMNs had about twice as many receptors as controls. Spinal motor neurons from a C9 knockout mouse, as well as motor neurons from patient postmortem tissue, had double the receptors as well. The reason for this glut is unclear, although C9ORF72 has been reported to affect vesicle trafficking, which may influence receptor movement (Farg et al., 2014; Aoki et al., 2017).
The motor neurons’ glutamate sensitivity matters, because ALS patients have high levels of glutamate in cerebrospinal fluid. Some researchers believe this is caused by DPRs that are taken up into astrocytes, where they interfere with splicing of an astrocytic glutamate transporter, rendering the glia unable to mop up extracellular glutamate (Lin et al., 1998; Aug 2014 news; Jun 2017 news; Aug 2017 news). Thus, loss of C9ORF72 may conspire with DPR toxicity to kill off motor neurons, the authors proposed.
In fact, glutamate sensitivity was specific to motor neurons. When the authors differentiated iPSCs from people with C9 expansions into dopaminergic neurons, those cells survived as well as controls in the presence of high glutamate levels. Ichida noted that motor neurons are believed to be more susceptible to excitotoxicity than other neurons due to poor calcium buffering (Van Den Bosch et al., 2000; Van Damme et al., 2007; Bogaert et al., 2010). Motor neurons selectively degenerate in ALS.
Excitotoxicity was not the only problem for C9ORF72-deficient iMNs. These cells also had about a third fewer lysosomes than controls. So did spinal motor neurons from the C9ORF72 knockout mouse. Expressing normal C9 in patient iMNs and C9 knockout iMNs restored lysosome numbers.
A lack of lysosomes might make motor neurons less able to degrade dipeptide repeat protein aggregates, the authors reasoned. In keeping with this idea, C9-deficient and patient iMNs cleared exogenous DPR deposits more slowly than control iMNs did, and the cells died faster. Conversely, adding C9ORF72 to patient iMNs lowered the number of DPR aggregates.
How might C9 control lysosome quantity? Ichida and colleagues found that about 80 percent of vesicle-bound C9ORF72 was located in early endosomes. There, it bound to the early endosomal marker, early endosome antigen 1. EEA1 in turn binds the lipid phosphatidylinositol 3-phosphate (PI3P) to drive endosomal maturation to lysosomes, as well as the fusion of those vesicles with autophagosomes. The findings suggested that a lack of C9 could block lysosomal maturation.
Previous studies have already implicated C9 in regulating autophagy (Webster et al., 2016; Sullivan et al., 2016). Some groups have found that stimulating autophagy boosts survival in ALS models, although others caution that more autophagy is not always better (May 2017 news; Sep 2017 news).
A Therapeutic Angle Emerges
The authors screened 800 small molecules for compounds to improve C9 iMN survival. Among the hits was an inhibitor of PIKFYVE, which localizes to early endosomes. PIKFYVE phosphorylates PI3P, converting it into an inactive form, thereby inhibiting endosomal fusion with lysosomes. Hence, inhibiting PIKFYVE stimulates autophagy. Intriguingly, the phosphatase FIG4, also found in early endosomes, opposes PIKFYVE function by activating PI3P. Loss-of-function mutations in FIG4 cause ALS, highlighting the importance of this pathway to the disease (Chow et al., 2009).
The PIKFYVE inhibitor rescued C9 patient iMN survival in a dose-dependent manner (see image above). The authors then tested a more potent and structurally distinct PIKFYVE inhibitor, apilimod. Apilimod has demonstrated safety in clinical trials for autoimmune disorders. It likewise rescued survival of C9 iMNs, and lowered their NMDA and AMPA receptor levels. In mouse models, apilimod blocked neurodegeneration and lowered DPR aggregates in hippocampal neurons.
“We are excited about this pathway because of the genetic link to the FIG4 form of ALS, and because PIKFYVE inhibition has the ability to rescue both the loss- and gain-of-function problems,” Ichida told Alzforum. He co-founded a company, AcuraStem in Monrovia, California, to develop PIKFYVE inhibitors for clinical trials. Company researchers have found that the body quickly metabolizes apilimod, perhaps explaining its failure in previous clinical trials. They are working on ways to stabilize the molecule. Baloh is an adviser to AcuraStem.
Shawn Ferguson at Yale University, New Haven, Connecticut, noted that the approved ALS drug riluzole inhibits excitotoxicity, and suggested comparing the effects of PIKFYVE inhibition to riluzole in cell cultures. “Significant follow-up is required to determine whether the beneficial effects observed by Shi et al. in relatively acute assays in very specific models can translate into significant therapeutic benefits in human disease that would exceed those seen with existing glutamate blockers such as riluzole,” Ferguson wrote.
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