Enhanced Autophagy Protects Against ALS Proteotoxicity

By boosting autophagy of misfolded superoxide dismutase 1, researchers protected motor neurons in vitro and in worms from this toxic protein encoded by an ALS gene. A small molecule called SecinH3 speeds up the flow of undesirable proteins through the lysosome, scientists from Children’s Hospital of Philadelphia report in the Jun 17 Journal of Neuroscience. It might prove beneficial in a variety of neurodegenerative diseases where altered proteins accumulate, speculated senior author Robert Kalb.

Neuronal Target.

SecinH3 interferes with the activity of cytohesins, managers of membrane trafficking labeled here in cultured neurons. [Courtesy of Zhai et al., The Journal of Neuroscience, 2015]

 

Kalb and colleagues were interested in how membrane trafficking participates in ALS pathogenesis. Both the endoplasmic reticulum and lysosomes have been implicated in the disease (see Mar 2009 News story; Sep 2009 News story; Jul 2014 News story). Joint first authors Jinbin Zhai, now at Temple University in Philadelphia, and Lei Zhang investigated the small ADP-ribosylation factors (ARFs) that manage many steps in moving membranes around the cell (reviewed in D’Souza-Schorey and Chavrier, 2006). ARFs hydrolyze GTP; their guanine nucleotide exchange factors, called cytohesins, remove the used-up GDP so ARFs can re-activate quickly. Mammals make at least six ARFs and four cytohesins. When researchers discovered that SecinH3 inhibits cytohesins (Hafner et al., 2006), the Kalb group was eager to test it out in mSOD1 model systems.

First, they infected cultured rat spinal motor neurons with a viral vector expressing mSOD1. Normally, this kills half the neurons within a week, but SecinH3 kept the cultures alive. The authors also observed neuroprotection when they transduced neurons with dominant-negative cytohesin mutants or interfering RNA against cytohesins.

Next, Zhai and Zhang tried blocking cytohesins in Caenorhabditis elegans expressing the mutant ALS gene. These mSOD1 worms swim slowly, but again, feeding them interfering RNA against cytohesins restored their speed and agility.

The authors suspected the treatments might work by removing misfolded SOD1. In primary neuronal cultures, SecinH3 reduced the concentration of misfolded SOD1, recognized with an antibody specific for the malformed protein, and insoluble, aggregated SOD1.

Based on these results, Zhai and Zhang tested whether SecinH3 treatment enhanced autophagy. They did so by quantifying LC3, an autophagosome component that gets constantly recycled. By stopping autophagy with a compound called Bafilomycin A, the researchers measured how much LC3 is going through the lysosome at a given time. When they treated HeLa or NSC34 cells with SecinH3, they observed more LC3, indicating more traffic on the autophagy pathway.

The researchers still have to work out which of the cell’s many cytohesins and ARFs are crucial to the autophagy process, and how inhibiting them speeds up protein disposal. However, the work could have far-reaching implications. Many other genes involved in ALS, such as profilin 1, also encode proteins that aggregate and might be cleared by cytohesin inactivation, pointed out Daryl Bosco of the University of Massachusetts Medical Center in Worcester, who was not involved in the study. Even in the sporadic form of the disease, some studies have suggested wild-type SOD1 folds improperly (see Oct 2010 News story). “The effect of SecinH3 does not appear specific to mutant SOD1, but rather to the downstream consequences of misfolded proteins,” Bosco said, “I would expect that their findings related to mutant SOD1 in the context of familial ALS are relevant and applicable to aberrantly modified wild type SOD1 in the context of sporadic ALS.”

Beyond ALS, membrane trafficking processes including endocytosis and autophagy are involved in Alzheimer’s, too, said Ralph Nixon of the Nathan Kline Institute in Orangeburg, New York, who was not involved in Kalb’s study. For example, ARF6 mediates the entry of BACE1 into cells (see Aug 2011 News story).  “The ability to upregulate autophagy certainly would apply to any disease where a misfolded or abnormal protein would be degraded through the autophagy pathway,” he said. “The big question is, at what level is this upregulation happening?” Lysosomes are impaired in both Alzheimer’s and Parkinson’s, Nixon pointed out, and without knowing how SecinH3 works, researchers cannot be sure if it would be able to fix the problems.

Kalb said scientists have not tried SecinH3 in other models of ALS or neurodegeneration. The compound itself would be no good as a human treatment because it was barely soluble in liquid, he said. Michael Famulok, a study co-author and developer of SecinH3, told Alzforum he mainly intended it as a research tool and has not made much effort to improve its solubility or develop derivatives for human use. The present study illustrates that inhibiting cytohesins in some way might be therapeutic, though, Kalb and colleagues wrote. Other scientists agreed. “They may be on to something very interesting,” commented Robert Brown of the University of Massachusetts Medical school, who did not participate in the study.—Amber Dance

Comments

    • User Profile Image Daryl Bosco
      University of Massachusetts Medical Center
    • Posted:

    I believe this work has implications for sporadic ALS, because the cytohesin inhibitor SecinH3 does not appear specific for mutant SOD1 but rather limits the downstream consequences of misfolded proteins, which may include ER-stress (based on their work). Researchers have identified aggregated and misfolded WT TDP-43 and WT SOD1 in postmortem tissues from sporadic ALS. It seems there is potential for clearance of these misfolded proteins by enhanced autophagic flux induced by SecinH3 (or inhibition/downregulation of cytohesins). Further, because aberrantly modified forms of WT SOD1 closely mimic mutant forms of SOD1 in many different cell-based toxicity assays, I would expect that their familial ALS findings are relevant/applicable to aberrantly modified WT SOD1 in the context of sporadic ALS.

    Also, because of the role of ADP ribosylation factors (ARF) in actin remodeling, I wonder whether modulation of ARF activity/function could be relevant to potential defects in actin dynamics induced by mutant profilin 1. Mutant profilin 1 also aggregates, as do most ALS-linked proteins, and therefore the mechanism involving autophagy induction could also apply here.

    View all comments by Daryl Bosco

  2. Genes involved in both innate immunity and autophagy—the natural process whereby cells remove defective organelles and infecting microrganisms—are being identified in studies of the genetics underlying several neurodegenerative diseases. In particular, it is rapidly becoming appreciated that the process of mitophagy, which is the specific removal of defective mitochondria, is important in Parkinson’s disease and ALS. The pertinent genes have been discovered by classical linkage genetics and genome-wide association studies and more recently exome sequencing has identified rarer genes in the same pathways—all of which have contributed to this excitement. For example, Parkin and PINK were discovered in Parkinson’s disease but seem to have a wider and important role in the orchestrated removal of aggregated proteins and organelles in both neurons and other cells. It is perhaps not surprising that neurons are particularly sensitive to the buildup of such cellular debris: They are long-lived cells that do not undergo mitosis, so the removal of “toxic waste” is likely to be of major importance.

    The process of autophagy and mitophagy is not simple. It involves protein ubiquitination and the proteasome, and the direction of aggregates and tagged mitochondria to the lysosome in very specific and subtle ways. PINK 1 and PARKIN, for example, polyubiquitylate defective mitochondria and target them to autophagosomes. The TANK binding kinase TBK1 phosphorylates, among other proteins, optineurin which is involved in autophagy. Both TBK 1 and optineurin are genes that have been implicated in ALS, which is primarily a motor neuron disease. This implies that the homeostasis of mitochondrial function is crucial to both dopaminergic and motor neurons and potentially other neurons as well.

     It is not just this connection that is provocative. There is data implicating superoxide dismutase (SOD), the first gene to be identified as causing a form of ALS, in autophagy. The SOD mutations causing ALS are thought to be gain of function. Moreover, as this article shows the ADP-ribosylation factor (ARF) GTPases involved in cytohesin function are important in vesicle trafficking, which is intimately involved in the process of autophagy. The inhibition of these GTPases has been shown to be “protective” in a C. elegans model of locomotor defects. Myosin VI and Rab GTPases (a related class of small GTPases) have also been heavily implicated in vesicle transport and autophagy. Whether these GTPases represent legitimate drug targets or not remains to be seen.

    It is interesting that less-complex organisms such as Drosophila and C. elegans are contributing to our understanding of these processes. For example, the use of modifier screens in Drosophila is complementing human genetic studies, because phenotypic modifiers can be identified genetically in flies, and then tested in the relevant mammalian cell biology systems to see how the homologous genes affect the process—as in cellular assays of mitophagy.

    Recent data suggests that drugs developed for targets that are genetically validated are at least twice as likely to enter clinical trials and beyond. This success is not just based on genes causing familial forms of disease but also encompasses genes, common alleles of which contribute to disease susceptibility. Putting this together with some of the observations above leads one to believe that in the complexity of neurodegeneration and the large number of potential targets that could be addressed, finding targets that modulate mitophagy will be a productive approach.

    View all comments by Tim Harris

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References

News Citations

  1. ER Struggles in Motor Neurons That Fall to ALS
  2. Research Brief: Mutant Cells Eat Mutant SOD1
  3. Can Autophagy Protect ALS Cell Models from Mutant TDP-43?
  4. Research Brief: SOD1 in Sporadic ALS Suggests Common Pathway
  5. Going It Alone—APP, BACE1 Take Distinct Routes to Endosome

Paper Citations

  1. D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol. 2006 May;7(5):347-58. PubMed.
  2. Hafner M, Schmitz A, Grüne I, Srivatsan SG, Paul B, Kolanus W, Quast T, Kremmer E, Bauer I, Famulok M. Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature. 2006 Dec 14;444(7121):941-4. PubMed.

External Citations

  1. ALS gene

Further Reading

Papers

  1. Cheung HN, Dunbar C, Mórotz GM, Cheng WH, Chan HY, Miller CC, Lau KF. FE65 interacts with ADP-ribosylation factor 6 to promote neurite outgrowth. FASEB J. 2013 Sep 20; PubMed.
  2. Shrivastava-Ranjan P, Faundez V, Fang G, Rees H, Lah JJ, Levey AI, Kahn RA. Mint3/X11gamma is an ADP-ribosylation factor-dependent adaptor that regulates the traffic of the Alzheimer's Precursor protein from the trans-Golgi network. Mol Biol Cell. 2008 Jan;19(1):51-64. PubMed.
  3. Miyazaki H, Yamazaki M, Watanabe H, Maehama T, Yokozeki T, Kanaho Y. The small GTPase ADP-ribosylation factor 6 negatively regulates dendritic spine formation. FEBS Lett. 2005 Dec 19;579(30):6834-8. PubMed.
  4. Ying H, Yue BY. Optineurin: The autophagy connection. Exp Eye Res. 2016 Mar;144:73-80. Epub 2015 Jul 2 PubMed.
  5. Karademir B, Corek C, Ozer NK. Endoplasmic reticulum stress and proteasomal system in amyotrophic lateral sclerosis. Free Radic Biol Med. 2015 Nov;88(Pt A):42-50. Epub 2015 Jun 12 PubMed.

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