Two new papers this month shore up the connection between cell waste disposal and function of neurons, and in the process, linking defects in the ubiquitin system to ataxia and to a form of intellectual disability. In the May 8 New England Journal of Medicine online, researchers trace Gordon Holmes syndrome, a rare condition characterized by ataxia, dementia, and reproductive defects, to mutations in a ubiquitin ligase and in a deubiquitinase. Another study, reported online May 16 in Molecular Cell, reveals that mutations in a ubiquitin-conjugating enzyme lead to intellectual disability by preventing proper disposal of mitochondria. That resembles the way mutations in the parkin gene, a genetic cause of familial parkinsonism, cause neurodegeneration (see ARF related news story). The two papers add to evidence that ubiquitination defects are not merely a symptom of neurological disease, but can lie at the root of degeneration.

“More and more diseases are now linked to the efficiency of the ubiquitin system,” commented Fred van Leeuwen of Maastricht University, the Netherlands, who was not involved with either study. Multiple genes implicate the ubiquitin pathway in amyotrophic lateral sclerosis (see ARF related news story) and Alzheimer’s disease (see ARF related news story). In the hopes of treating neurodegeneration, researchers are pursuing therapies to boost the ubiquitin-proteasome degradation system and promote removal of pathological proteins (see ARF related news story).

Ubiquitin Genes Link Ataxia, Reproductive Defects
Men and women with Gordon Holmes syndrome typically fail to go through puberty normally, or they develop reproductive problems such as infertility in early adulthood. The disease is rooted in a lack of gonadotropic-releasing hormone (GnRH), which neurons in the pituitary normally produce. Although GnRH treatment could potentially help some people, no good therapies for this disease exist, said Stephanie Seminara of Massachusetts General Hospital in Boston, senior author of the NEJM paper.

The disease also causes ataxia, typically after the reproductive symptoms surface. Patients’ gait becomes clumsy and their speech slurred. Joint first authors David Margolin and Yee-Ming Chan and colleagues report that some people with the syndrome also exhibit the memory loss and personality changes typical of dementia, though more patients need to be studied before this dementia can be fully characterized.

To find genes involved in Gordon Holmes syndrome, the researchers sequenced the exomes of three siblings from an affected Middle Eastern kindred. Several of the married couples in the family are cousins. Margolin and Seminara have been working with this extended family for more than a decade (Seminara et al., 2002). Their results pointed to two candidate genes: the E3 ubiquitin ligase ring finger protein 216 (RNF216) and the deubiquitinase ovarian tumor domain containing 4 (OTUD4). E3 ligases—parkin among them—join up with E2 ubiquitin-conjugating enzymes to label unwanted proteins with ubiquitin. By removing the tags, deubiquitinases can spare proteins from degradation. They also remove ubiquitin from proteins as they enter the proteasome, preserving the tag for future use. Scientists know little about OTUD4’s biological role.

All three siblings were homozygous for one missense mutation in RNF216 and one in OTUD4. Seminara was unable to deduce which gene caused the disease based on the genotype of family members alone, since no affected person was homozygous for either one of the mutations. She turned to joint senior author Nicholas Katsanis and joint first author Maria Kousi, at Duke University in Durham, North Carolina, to examine the function of each of the two genes in a zebrafish model.

Knocking down either RNF216 or OTUD4 resulted in fish embryos with modestly disorganized cerebellums. Blocking both caused a more severe phenotype. Normal human versions of each gene rescued the defects, but genes containing the same mutations as the Middle Eastern family did not. Katsanis concluded that both genes contributed to the disease, and that their proteins somehow interact with each other. The authors suspect that the missense mutations result in partially functional OTUD4 and RNF216 protein, and together these two hits debilitate the ubiquitin pathway.

Seminara confirmed the role of RNF216 in a handful of people from other Gordon Holmes syndrome families. She discovered five more people with novel RNF216 mutations. The OTUD4 gene appeared normal. One person carried two different mutations, leading to early termination of both copies of the RNF216 transcript. This person likely had no RNF216 function, leading to the disease in the absence of OTUD4 mutations, the authors surmised. Four more volunteers possessed early termination or missense mutations on only one RNF216 allele. These people inherited the mutant allele from an unaffected parent. Perhaps, the authors posit, the children have a second, unknown genetic hit that works with the RNF216 haploinsufficiency to cause Gordon Holmes syndrome.

Interestingly, the authors, specifically linked RNF216 mutations to dementia. These five individuals, plus the three affected members of the Middle Eastern kindred, developed dementia in addition to the other symptoms. In contrast, people with Gordon Holmes syndrome but normal RNF216 showed no signs of dementia.

How these mutations lead to these different phenotypes remains murky. The researchers observed ubiquitinated inclusions in the brain of one sibling after she died. Katsanis suggested that specific protein targets of RNF216 and OTUD4 might accumulate in the brain. Alternatively, the mutations might upset protein homeostasis overall, altering ubiquitination of many proteins, he speculated.

The authors are also unable to explain why the disease targets the GnRH-producing cells in the pituitary along with certain cerebellar and hippocampal neurons. “Their finding makes me wonder if GnRH deficiency might contribute to the neurodegenerative changes,” wrote Dongsheng Cai of the Albert Einstein College of Medicine in the Bronx, New York, in an e-mail to Alzforum. Cai, who was not involved in the work, recently reported that a decline in GnRH production accelerates aging (see ARF related news story on Zhang et al., 2013). Cai found that NF-κB, a known target of RNF216, can shut down GnRH secretion. He speculated that if RNF216 fails to ubiquitinate NF-κB, then the transcription factor could accumulate sufficiently to suppress GnRH production. Katsanis said he plans to examine NF-κB activity in zebrafish and in cell culture models of RNF216 deficiency.

Faulty Mitophagy in Intellectual Disability
A better handle on the role of ubiquitination in disease comes from the Molecular Cell paper. First author Dominik Haddad and joint senior authors Vanessa Morais and Patrik Verstreken of the VIB Center for the Biology of Disease in Leuven, Belgium, studied X-linked intellectual disability (XLID), a clinically and genetically diverse condition. Children with XLID manifest low intellectual ability and poor adaptive skills before the age of 18. One form of XLID results from mutations in another E2 ubiquitin ligase, the ubiquitin-conjugating enzyme E2A (UBE2a; Nascimento et al., 2006). Using X-exome sequencing, Haddad and colleagues identified two families harboring pathogenic mutations in this gene. Lymphocytes from affected male children made much less of the protein than normal.

Knocking out the UBE2a homologue in fruit flies caused neurons to fill up with clumps of mitochondria. Haddad and colleagues found that these mitochondria failed to maintain the proper charge across their membranes. The knockout flies died before adulthood, but replacing the UBE2a gene kept them alive. The researchers performed the same experiments in fibroblasts from UBE2a knockout mice and in lymphocytes from patients, seeing similar mitochondrial defects.

This phenotype reminded Verstreken of defects in mitochondrial autophagy, or mitophagy, caused by parkin mutations. Parkin’s role in mitophagy is well established. The researchers hypothesized that as an E3 ligase, parkin might join up with the E2 UBE2a to promote degradation of sickly mitochondria. This would mirror parkin’s interaction with PINK1 in Parkinson’s disease. Indeed, when the team treated mouse embryonic fibroblasts with a compound to depolarize mitochondria and trigger mitophagy, UBE2a and parkin coimmunoprecipitated. By fluorescence microscopy, the researchers confirmed that parkin labeled with green fluorescent protein localized to mitochondria under the same conditions. They also performed in-vitro experiments showing that UBE2a facilitated the formation of ubiquitin chains by parkin, as an E2 enzyme ought to do for its E3 partner.

The team concluded that UBE2a and parkin likely work together, as parkin does with PINK1, to promote mitophagy. As for the targets of their ubiquitinating activity, Verstreken said mitofusin, which PINK1 and parkin ubiquitinate, would be his best guess. Despite this molecular similarity, clinically the intellectual disability shares no features with Parkinson’s, Verstreken said. However, he speculated that affected people might die too young—typically in their thirties—to develop parkinsonian symptoms.—Amber Dance


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News Citations

  1. Abnormal Mitochondrial Dynamics—Early Event in AD, PD?
  2. New ALS Genes Implicate Protein Degradation, Endoplasmic Reticulum
  3. The Ubiquitin Proteasome System—Out of STEP in AD?
  4. Therapeutic Approaches Target Deubiquitinase, Protein Turnover
  5. Do Microglia in the Hypothalamus Drive Aging?

Paper Citations

  1. . Hypogonadotropic hypogonadism and cerebellar ataxia: detailed phenotypic characterization of a large, extended kindred. J Clin Endocrinol Metab. 2002 Apr;87(4):1607-12. PubMed.
  2. . Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature. 2013 May 1; PubMed.
  3. . UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked mental retardation syndrome. Am J Hum Genet. 2006 Sep;79(3):549-55. PubMed.

Further Reading


  1. . Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8638-43. PubMed.
  2. . Targeting ubiquilin-1 in Alzheimer's disease. Expert Opin Ther Targets. 2013 Jul;17(7):795-810. PubMed.
  3. . β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1. J Biol Chem. 2013 Jun 7;288(23):16937-48. PubMed.
  4. . Accumulation of the parkin substrate, FAF1, plays a key role in the dopaminergic neurodegeneration. Hum Mol Genet. 2013 Apr 15;22(8):1558-73. PubMed.
  5. . Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca(2+) transfer to sustain cell bioenergetics. Biochim Biophys Acta. 2013 Apr;1832(4):495-508. PubMed.
  6. . Tau degradation: The ubiquitin-proteasome system versus the autophagy-lysosome system. Prog Neurobiol. 2013 Jun;105:49-59. PubMed.
  7. . Proteostasis and neurodegeneration: The roles of proteasomal degradation and autophagy. Biochim Biophys Acta. 2013 Mar 21; PubMed.
  8. . The E3 ligase parkin maintains mitochondrial integrity by increasing linear ubiquitination of NEMO. Mol Cell. 2013 Mar 7;49(5):908-21. PubMed.

Primary Papers

  1. . Ataxia, dementia and hypogonadism caused by disordered ubiquitination. N Engl J Med.. 2013 May;
  2. . Mutations in the Intellectual Disability Gene Ube2a Cause Neuronal Dysfunction and Impair Parkin-Dependent Mitophagy. Mol Cell. 2013 May 14; PubMed.