The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

Abstract

Proper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability (XLID). Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

“… 27 , 28 , 29 , 30 The stability of Dcx is post-translationally regulated by UPS, which is mediated by E3 ligases, including CRL3 and Mdm2. 13 , 31 Therefore, we hypothesized that CRL4 might also control Dcx stability. Through immunoblotting following immunoprecipitation, we confirmed that a small fraction of Dcx interacts with Cul4a and Cul4b in DIV4 neurons ( Figure 8 A).…”

“… 11 , 12 Similarly, Kelch-like 15 (Klhl15), a substrate adaptor for CRL3 implicated in X-linked intellectual disability, promotes destabilization of the Dcx protein, thereby inhibiting dendrite outgrowth. 13 Mutations in human CUL4B , which encodes cullin 4b in CRL4, are also linked to X-linked intellectual disability. 14 , 15 Cul4b knockout mice exhibit spatial learning defects and epileptic hypersensitivity with abnormal dendritic morphology in the interneurons of the hippocampus.…”

Cullin-RING E3 ubiquitin ligase 4 controls axonal morphogenesis during neuronal development

“…Mdm2 restricts the level of Dcx via the ubiquitinationproteasome degradation system, thereby inhibiting dendritic spine morphogenesis in olfactory bulb interneurons 32 . Klhl15-CRL3 complex targets Dcx, resulting in restriction of neuritogenesis 12 . This implies that CRL3 and CRL4 may share substrates and have overlapping functions in neuritogenesis.…”

“…It has been well-known that Dcx facilitates axon and dendrite morphogenesis during neurodevelopment by stabilizing microtubules and regulating actin laments [28][29][30][31] . And Dcx protein is post-translationally regulated by the ubiquitination-proteasome system mediated by CRL3 or Mdm2 E3 ligase 12,32 . Therefore, we postulated that CRL4 may also control stability of Dcx among the CRL4-interacting proteins.…”

“…Genetic depletion of the Cul3 in CRL3 induces abnormal accumulation of cytoskeletal proteins involved in neuronal migration and dendritogenesis, thereby leading to social and cognitive impairments implicated with autism spectrum disorder 10,11 . Similarly, Kelch-like 15 (Klhl15), a substrate adaptor of CRL3 implicated in X-linked intellectual disability, promotes destabilization of the Dcx protein, thereby inhibiting dendrite outgrowth 12 . Meanwhile, genetic mutations in CUL4B, a scaffold protein of CRL4, are linked to Xlinked intellectual disability 13,14 .…”