Mammalian TRIM67 Functions in Brain Development and Behavior

Abstract

Class I members of the tripartite motif (TRIM) family of E3 ubiquitin ligases evolutionarily appeared just prior to the advent of neuronal like cells and have been implicated in neuronal development from invertebrates to mammals. The single Class I TRIM in Drosophila melanogaster and Caenorhabditis elegans and the mammalian Class I TRIM9 regulate axon branching and guidance in response to the guidance cue netrin, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Opitz Syndrome, characterized by midline defects and often intellectual disability. We find that although TRIM67 is the least studied vertebrate Class I TRIM, it is the most evolutionarily conserved. Here we show that mammalian TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC and is differentially enriched in specific brain regions during development and adulthood. We describe the anatomical and behavioral consequences of deletion of murine Trim67. While viable, mice lacking Trim67 exhibit abnormal anatomy of specific brain regions, including hypotrophy of the hippocampus, striatum, amygdala, and thalamus, and thinning of forebrain commissures. Additionally, Trim67−/− mice display impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function, and sensorimotor gating, whereas several other behaviors remain intact. This study demonstrates the necessity for TRIM67 in appropriate brain development and behavior.

TRIM9 and TRIM67 Are New Targets in Paraneoplastic Cerebellar Degeneration

“…No staining was observed on double KO mice brain slices (Figure 2B), indicating that the Abs in the CSF from patient 1 were only raised against TRIM9 and TRIM67. Immunostaining on Trim9 −/− single KO mouse brain found expression of TRIM67, and on Trim67 −/− mouse brain found expression of TRIM9 (Figure 2B); TRIM9 was widely expressed, notably in the hippocampus, and TRIM67 in the cerebellum, as previously described [6]. …”

“…Immunostaining of Trim67 −/− mouse brain confirmed these observations. TRIM67 is abundantly expressed in the cerebellum of both mouse and human [6,11], and immunostaining with patient CSF herein showed a similar pattern.…”

“…Furthermore, in animal models, absence of TRIM9 causes excessive dendrite arborization, mislocalization and decreased dendritic density in adult born neurons of the dentate gyrus, leading to impairment of learning and memory [18]. TRIM67 interacts with TRIM9 and DCC and plays important role in brain development and behavior, including learning and memory and cognitive flexibility [6]. These two proteins seem therefore to play a major role in neuronal function, and we therefore investigated the pathogenicity of Abs on neurons.…”

“…Timed pregnant females were obtained by placing male and female mice together overnight; the following day was designated as E0.5 if the female had a vaginal plug. Trim9 −/− mice and Trim67 −/− mice have been previously described [5,6]. Trim9 −/− : Trim67 −/− mice, generated by crossing Trim9 −/− mice and Trim67 −/− mice, were viable and will be described in more detail below.…”

“…To confirm the target of the Abs, HEK 293 cells were transfected with pCS2- Trim9 (mouse) plasmid [8] or pCS2- Trim67 (mouse) plasmid [6] for transient overexpression. Fixed and permeabilized cells were then immunostained using patient CSF (1:100) and commercial mouse monoclonal anti-Myc antibody (ab9106, Abcam, Paris, France) and then they were revealed with appropriate fluorochrome-conjugated secondary antibodies (1:1500, A11013, and A11001, Thermofischer, Courtaboeuf, France).…”