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The GEF Trio and Its Role in Autism Spectrum Disorders

Intellectual disabilities (e.g., neurodevelopmental disorders, autism spectrum disorders [ASDs]) are associated with abnormal development of dendrites and dendritic spines. ASDs are a complex set of behaviorally defined disorders, characterized by impairments in social interaction, communication, and restricted or stereotyped behaviors. Recent studies estimate that 1% of the world-wide population has an ASD1. Dendritic spines are comprised of F-actin and the structural and functional plasticity of spines depend upon the dynamic regulation of actin by Rho-family GTPases. Indeed, Rac and PAK effector proteins are essential regulators of normal brain development and function, including dendritic spine initiation, elongation, and branching2-4. Recent genetic studies revealed that individuals with intellectual disabilities express mutated versions of genes involved in Rho-family GTPase signaling such as a Rho-family GTPase activating protein (GAP), the serine/threonine kinase PAK3, and the Rac/Cdc42 guanine exchange factor (GEF) aPIX5. In addition, the PAK inhibitor FRAX486 is an effective treatment for fragile X syndrome (FXS), the most common inherited form of autism and cognitive disability. FRAX486 reversed dendritic spine and behavioral abnormalities in an in vivo model of FXS6. Moreover, Rac1 activation or inhibition of cofilin, an actin depolymerizing protein, rescues ASD-like phenotypes in Shank3 knock-out mice, an in vivo model of ASDs7-12.

The Rho-family GEF Trio is a multi-functional, multi-domain GEF widely expressed throughout the developing brain13-15 (Fig. 1). Trio’s highest level of neuronal expression is in the late prenatal/early postnatal period, suggesting a role in early neuron development13-15. Multiple isoforms of Trio exist with a regional-, developmental-, and isoform-specific expression pattern (Fig. 1). In hippocampal and cortical neurons, Trio-9 is the predominant isoform13,14. Trio mediates activation of the Rho-family GTPases RhoA and Rac1 through two distinct GEF domains, both of which regulate a multitude of actin-based structural changes important in neurotransmission16-17.

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Figure 1: ASD-related mutations in the Rho-family GEF Trio. A. Missense mutations across multiple domains; B. 16 exon deletion resulting in loss of entire GEF1 domain; C. Single nucleotide deletion resulting in formation of stop codon in GEF1/DH1 domain. For each mutation, the cognitive disorder diagnosis is provided. Trio’s amino acid sequence is from NP_009049.2.

This newsletter focuses on Trio-mediated activation of Rac1 and the presumed Rac1-mediated remodeling of the F-actin cytoskeleton necessary for subsequent changes in excitatory glutamatergic neurotransmission on hippocampal and cortical dendritic spines. Dysfunctional glutamatergic neurotransmission is implicated in ASDs18-21. Recent studies have begun to examine the relationship between Trio, Rac1, glutamatergic neurotransmission, and ASDs. Overexpression of wild-type Trio-9 in neurons of hippocampal slice cultures increases AMPA receptor (AMPAR)-mediated evoked excitatory post-synaptic currents (eEPSCs) and Trio knockdown produced the opposite effect22. Importantly, NMDA receptor (NMDAR)-mediated synaptic activity was not significantly affected by either treatment22. Conversely, Trio knockdown by transfected shRNAs produced a selective increase in AMPAR-mediated eEPSCs in neurons of hippocampal slice cultures23,24. Trio knockdown also promoted dendritic outgrowth and branching, as well as decreasing AMPAR endocytosis in primary hippocampal cultures23,24. These differential effects are likely due to the developmental time point at which Trio expression was reduced.

Building on this understanding of Trio/Rac1-mediated regulation of glutamatergic neurotransmission and its role in ASDs, researchers examined how Trio mutations affect its activities in ASD models. Within the Rac GEF-domain of the Trio gene, several mutations result in reduced Rac1 activation and are associated with an inherited developmental delay phenotype that displays as microcephaly, as well as intellectual disability and dysmorphism with digital features25 (Fig. 1). Furthermore, within Trio’s Rac activation subdomain (GEF1/DH1; 175 amino acids) of individuals with ASDs or ASD-related disorders, there are 6 de novo missense mutations and a single nucleotide deletion that results in the formation of a stop codon (Fig. 1). Depending on the mutation, Trio can be either hypo- or hyperfunctional in regards to activating Rac1 and glutamatergic neurotransmission in in vitro models of ASDs. Trio mutations resulting in hypoactivity reduced Rac1 activation and AMPAR-mediated eEPSCs due to an increased number of silent synapses (i.e., synapses devoid of AMPARs) with no change in NMDAR-mediated eEPSCs. Mutations producing hyperfunctionality triggered greater Trio-activated Rac1 levels and an increase in AMPAR and NMDAR-mediated eEPSCs due to increased glutamatergic synaptogenesis26. Both Trio/Rac1-mediated under- and over-stimulation of glutamatergic neurotransmission has adverse neurodevelopmental and/or cognitive effects that must be accounted for in diagnosing and treating ASDs and related disorders.

Summary

Recent data strongly suggest that mutant Trio-mediated bidirectional dysregulation of Rac1-mediated re-organization of the actin cytoskeleton at glutamatergic synapses is a hallmark of ASDs. In this way, Trio/Rac1 signaling could serve as a point of confluence and potential molecular mechanism underlying the behavioral and cognitive deficits associated with ASDs. To help researchers better understand the role of Rho-family GTPases in regulating actin in ASDs, neurodevelopmental disorders, and intellectual disabilities, Cytoskeleton, Inc., offers purified GEFs and activation assays for small GTPases, as well as activators, inhibitors, and antibodies. Additional reagents include live cell imaging probes for F-actin and kits to study actin polymerization, binding, and in vivo levels of monomeric vs polymeric actin.

Related Products & Resources

GTPase Assay Kits

RhoGEF Exchange Assay (Cat. # BK100)

Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK035)

Rac1 G-LISA Activation Assay (Luminescence format) (Cat. # BK126)

Rac1 G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK128)

Spirochrome™ Live Cell Imaging Probes

Spirochrome SiR-Actin Kit (Cat. # CY-SC001)

GEF Proteins

ARNO GEF Protein (Sec7 Exchange Domain, aa31-267, 6xHis tag) (Cat. # CS-GE07)

Dbs protein GEF domain: His tagged: human recombinant (Cat. # GE01-A)

RasGRF1 GEF Protein (Cdc25 Exchange Domain, aa1038-1270, MBP tag) (Cat. # CS-GE03)

SOS1 Protein (ExD Exchange Domain, aa564-1049, 6xHis tag) (Cat. # CS-GE02)

Tiam1 GEF Protein (DHPH Exchange Domain, aa1040-1406, MBP tag) (Cat. # CS-GE04)

Vav1 GEF Protein (DHPHC1 Exchange Domain, Y174D mutant, aa168-522, 6xHis tag) (Cat. # CS-GE05)

Vav2 GEF Protein (DH Exchange Domain, aa189-374, 6xHis tag) (Cat. # CS-GE06)

References

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  2. Boda B. et al. 2004. The mental retardation protein PAK3 contributes to synapse formation and plasticity in hippocampus. J. Neurosci. 24, 10816-10825.
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  4. Huang W. et al. 2011. P21-activated kinases 1 and 3 control brain size through coordinating neuronal complexity and synaptic properties. Mol. Cell Biol. 31, 388-403.
  5. Ramakers G.J.A. 2002. Rho proteins, mental retardation and the cellular basis of cognition. Trends Neurosci. 25, 191-199.
  6. Dolan B.M. et al. 2013. Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486. Proc. Natl. Acad. Sci. U.S.A. 110, 5671-5676.
  7. Bonaglia M.C. et al. 2001. Disruption of the ProSAP2 gene in a t(12;22)(q24.1;q13.3) is associated with the 22q13.3 deletion syndrome. Am. J. Hum. Genet. 69, 261–268.
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  17. Chhatriwala M.K. et al. 2007. The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. J. Mol. Biol. 368, 1307-1320.
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  19. Rojas D.C. 2014. The role of glutamate and its receptors in autism and the use of glutamate receptor antagonists in treatment. J. Neural. Transm. 121, 891-905.
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  21. Bourgeron T. 2015. From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat. Rev. Neurosci. 16, 551-563.
  22. Herring B.E. and Nicoll R.A. 2016. Kalirin and Trio proteins serve critical roles in excitatory synaptic transmission and LTP. Proc. Natl. Acad. Sci. U.S.A. 113, 2264-2269.
  23. Ba W. et al. 2016. TRIO loss of function is associated with mild intellectual disability and affects dendritic branching and synapse function. Hum. Mol. Genet. 25, 892-902.
  24. Ba W. and Kasri N.N. 2017. RhoGTPases at the synapse: An embarrassment of choice. Small GTPases. 8, 106-113.
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