Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth

Reviewer #1 (Public Review):

Summary:

In this work, Frank, Bergamasco, Mlodzianoski et al study two microcephaly-associated patient variants in TRABID to identify and characterize a previously unrecognized role of this deubiquitylation enzyme during neurodevelopment. The authors generate TRABID p.R438W and p.A451V knock in mice, which exhibit smaller neuronal and glial cell densities as well as motor deficits, phenotypes that are consistent with the congenital defects observed in the patients. Through in vitro and cellular immunoprecipitation assays, the authors demonstrate that the p.R438W variant impairs the K29- and K63-chain cleavage activity of TRABID, while the p.A451V variant reduces binding to the STRIPAK complex, a previously identified TRABID interactor with established functions in cytoskeletal organization and neural development. Ubiquitylation assays performed in HEK293T cells further reveal that the hypomorphic patient variants are deficient in deubiquitylating APC, a previously identified substrate of TRABID that has been shown to control the neuronal cortical cytoskeleton during neurite outgrowth. Ex vivo experiments provide evidence that axonal APC trafficking and neurite outgrowth is disturbed in differentiating neural progenitors isolated from mouse embryos carrying Trabid patient alleles. From these experiments the authors propose a model in which TRABID- and STRIPAK-dependent APC deubiquitylation regulates its axonal trafficking to ensure faithful neurite outgrowth and misregulation of this function leads to neurodevelopmental phenotypes in TRABID/ZRANB1 patients.

Strengths:

This study describes a previously unrecognized function of TRABID in neurodevelopment and establishes knock in mice as model to study congenital defects of TRABID/ZRANB1 patients. In addition, the authors identify control of axonal trafficking of APC by deubiquitylation as a potential mechanism through which TRABID regulates neurite outgrowth and whose dysregulation could be the molecular basis of the neurodevelopmental phenotypes observed in TRABID/ZRANB1 patients.

Weaknesses:

While the proposed underlying mechanism of how hypomorphic TRABID mutations lead to the patient phenotypes is conceivable and supported by the author's data, there is no functional evidence provided that the mouse phenotypes (reduced neuron/glia densities or motor deficits) are indeed due to aberrant APC deubiquitylation and trafficking. In addition, some aspects of the proposed mechanism, i.e. the claim that APC deubiquitylation is STRIPAK-dependent, should be strengthened by orthogonal approaches.

Reviewer #2 (Public Review):

Summary:

Although Trabid missense mutations are identified across a range of neurodevelopmental disorders, its role in neurodevelopment is not understood. Here the authors study two different patient mutations and implicate defects in its deubiquitylating activity and interactions with STRIPAK. Knockin mice for these mutations impaired trafficking of APC to microtubule plus ends, with consequent defects in neuronal growth cone and neurite outgrowth.

The authors focus on R438W and A451V, two missense mutations seen in patients. Recombinant fragments showed R438W is nearly completely DUB-dead whereas A451V showed normal activity but failed to efficiently precipitate STRIPAK. Knockin of these mutations showed a partially penetrant reduced cortical neuronal and glial cell numbers and reduced TH+ neurons and their neuronal processes. Cell culture demonstrated that both DUB and STRIPAK-binding activities of Trabid are required for efficient deubiquitylation of APC in cells, and alter APC transport along neurites. APC-tdTomato fluorescent reporter mice crossed with the Trabid mutants confirmed these results. The results suggest that Trabid's mechanism of action is to suppress APC ubiquitylation to regulate its intracellular trafficking and neurite formation.

Strengths:

Solid manuscript with in vivo and in vitro demonstration of mechanism of action

Weaknesses:

Much of the work relies on prior discoveries of Trabid's role in STRIPAK and APC related functions, so the novelty is somewhat reduced.