Gordon Holmes Syndrome Model Mice Exhibit Alterations in Microglia, Age, and Sex-Specific Disruptions in Cognitive and Proprioceptive Function.

Gordon Holmes syndrome (GHS) is a neurological disorder associated with neuroendocrine, cognitive, and motor impairments with corresponding neurodegeneration. Mutations in the E3 ubiquitin ligase RNF216 are strongly linked to GHS. Previous studies show that deletion of Rnf216 in mice led to sex-specific neuroendocrine dysfunction due to disruptions in the hypothalamic-pituitary-gonadal axis. To address RNF216 action in cognitive and motor functions, we tested Rnf216 knock-out (KO) mice in a battery of motor and learning tasks for a duration of 1 year. Although male and female KO mice did not demonstrate prominent motor phenotypes, KO females displayed abnormal limb clasping. KO mice also showed age-dependent strategy and associative learning impairments with sex-dependent alterations of microglia in the hippocampus and cortex. Additionally, KO males but not females had more negative resting membrane potentials in the CA1 hippocampus without any changes in miniature excitatory postsynaptic current (mEPSC) frequencies or amplitudes. Our findings show that constitutive deletion of Rnf216 alters microglia and neuronal excitability, which may provide insights into the etiology of sex-specific impairments in GHS.

The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons.

Of the hundreds of E3 ligases found in the human genome, the RING-between RING (RBR) E3 ligase in the LUBAC (linear ubiquitin chain assembly complex) complex HOIP (HOIL-1-interacting protein or RNF31), contains a unique domain called LDD (linear ubiquitin chain determining domain). HOIP is the only E3 ligase known to form linear ubiquitin chains, which regulate inflammatory responses and cell death via activation of the NF-κB pathway. We identified an amino acid sequence within the RNF216 E3 ligase that shares homology to the LDD domain found in HOIP (R2-C). Here, we show that the R2-C domain of RNF216 promotes self-assembly of all ubiquitin chains, with a dominance for those assembled via K63-linkages. Deletion of the R2-C domain altered RNF216 localization, reduced dendritic complexity and changed the distribution of apical dendritic spine morphology types in primary hippocampal neurons. These changes were independent of the RNF216 RBR catalytic activity as expression of a catalytic inactive version of RNF216 had no effect. These data show that the R2-C domain of RNF216 diverges in ubiquitin assembly function from the LDD of HOIP and and functions independently of RNF216 catalytic activity to regulate dendrite development in neurons.