Defects in translational regulation contributing to human cognitive and behavioral disease

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Recent data suggest that the levels of many synaptic proteins may be tightly controlled by the opposing processes of new translation and protein turnover in neurons. Alterations in this balance or in the levels of such dosage-sensitive proteins that result in altered stoichiometry of protein complexes at developing and remodeling synapses may underlie several human cognitive diseases including Fragile X Syndrome, autism spectrum disorders, Angelman syndrome and non-syndromic mental retardation. While a significant amount is known about the transduction of membrane signals to the translational apparatus through kinase cascades acting on general translation factors, much less is understood about how such signals may influence the activity of mRNA-specific regulators, their mechanisms of action and the specific sets of mRNAs they regulate. New approaches to the unbiased in vivo identification of maps of binding sites for these proteins on mRNA is expected to greatly increase our understanding of this crucial level of regulation in neuronal development and function.

Highlights

► Activity-dependent translation is required for proper synaptic development and plasticity. ► Specific mRNA-binding proteins including FMRP may regulate such translation in neurons. ► New methods are being used to identify physiologically relevant mRNA targets of RNABPs.

Introduction

Normal human cognition is dependent on the proper wiring of the central nervous system during critical periods in development, as well as the maintenance and plasticity of this network in response to experience and insult throughout life. Communication between neurons allows the formation and fine-tuning of neuronal connections to coordinate cellular activity into circuits. A fundamental unit of communication in neuronal networks is the synapse. Synapses are comprised of a relatively well-defined set of proteins many of which function in multi-protein complexes. As such, they may be present in defined stoichiometric ratios arising in some cases from the coordinated synthesis, packaging and delivery of ‘units’ of these multi-protein complexes to axons or dendrites where new or modified synapses are needed. One example of such a cellular strategy for achieving proper stoichiometry is the set of presynaptic scaffolding proteins including bassoon, piccolo, RIM and munc13 [1, 2]. These proteins are synthesized in the neuronal cell body and transported down the axon of the presynaptic cell in ‘piccolo-bassoon transport vesicles’ or PTVs. In response to signals enticing formation of a new synapse, one or more of these quanta of synaptic proteins is inserted into the presynaptic membrane [1].

Perhaps owing to these stoichiometric constraints, alterations in the functional levels of several synaptic proteins are believed to underlie defects in cognition and behavior in human disease. Autism is one example; haplo-insufficiency of Shank3, neurexins, or neuroligins can cause the disease [3, 4]. Shank3 is a scaffolding protein in the postsynaptic density (or PSD, the assembly of postsynaptic proteins of excitatory glutamatergic synapses), and is believed to be a key organizer of the PSD as a component of defined multi-protein complexes [5, 6]. Similarly, haplo-insufficient mutations in neurexins and neuroligins, presynaptic and postsynaptic cell adhesion molecules mediating synapse formation and stabilization, have been linked to autism spectrum disorders (ASDs), Tourette's syndrome, schizophrenia and nonspecific learning disabilities [7].

Variation in protein expression levels can also arise in individuals owing to de novo microdeletions and microduplications, giving rise to one or three alleles of a gene rather than the usual two (referred to as copy number variations; CNVs) [8]. CNVs have been shown to be much more common than expected; as many as one in eight births harbors a microdeletion and one in fifty, a microduplication [9]. Several large-scale studies in human copy number variation have examined the relationship of such events with cognitive diseases such as the ASDs and schizophrenia [10]. A fascinating conclusion from these studies is that 50% increases in levels of certain dosage-sensitive synaptic proteins is linked to cognitive diseases as well as the more commonly appreciated 50% decreases arising from loss-of-function mutations. Interestingly, many of the individual synaptic proteins whose dysregulation or mutation is related to the ASDs have now been linked to disease through both underexpression and overexpression. Again, Shank3 is a good example; duplication of the 22q13 region encompassing the Shank3 gene has been linked to severe impairment of social communication [4]. This and similar examples can explained by the gene balance hypothesis, which posits that deleterious phenotypes can arise from underexpression or overexpression of the same dosage-sensitive proteins because either can disrupt the stoichiometry of the same complex [11, 12, 13]. In sum, this evidence supports the concept that the expression levels of many synaptic proteins are crucial to the formation and maintenance of proper synaptic function.

The expression level of many synaptic proteins may be tightly controlled by the balance between translation and turnover. The growing number of developmental cognitive diseases whose underlying cause is a defect in the regulation of either translation or turnover suggests that the equilibrium between these opposing processes is a sensitive point in establishing normal cognition and behavior. The first such disease to be characterized was Fragile X Syndrome, caused by a triplet repeat expansion that silences expression of the Fragile X Mental Retardation protein, FMRP, thought to repress neuronal activity-dependent translation [14]. Subsequently, some cases of autism were found to be caused by mutations in PTEN, TSC2 and NF1, three proteins with a shared function to repress the mammalian target of rapamycin (mTOR) pathway that is important for activity-dependent initiation of new translation [15]. On the side of protein turnover, both Angelman syndrome and X-linked syndromic mental retardation have been linked to defects or alterations in expression of the ubiquitin ligases UBE3A and HUWE1 respectively [16, 17, 18] that mark proteins for degradation by the proteasome. Taken together, a compelling argument can be made that elucidating the mechanisms regulating neuronal protein translation and turnover are likely to shed light on fundamental aspects of human cognition and neuronal function. This review is focused primarily on the function and regulation of neuronal translation, and the reader is directed to excellent reviews on synaptic activity-regulated protein turnover for a complementary view of this equilibrium [19•, 20].

Section snippets

Mechanisms for translational regulation in neurons

While most cells have the ability to alter translation in response to environmental signals neurons have an additional need for specific mechanisms of translational control because of their architecture (Figure 1). This is partly due to a need for spatial control, exemplified by the ability of local groups of synapses to alter their ‘strength’ in response to local input using mechanisms dependent on new protein synthesis in the processes rather than cell body. There is an additional need for

FMRP: an example of an mRNA-specific regulatory protein

Perhaps the most appealing approach to identifying the most relevant mRNA-specific translation factors is to mine the documentation of naturally occurring human mutations in neuronal RNABPs whose function is likely to include translational control and that are causally linked to cognitive and behavioral symptoms. Fragile X Syndrome, characterized by intellectual disability, autistic symptoms, and childhood seizures is a model example. In Fragile X patients, a CGG triplet repeat expansion in the

Challenges inherent in the identification of sets of regulated mRNAs

While identification of important neuronal regulators of translation (illustrated above by work on FMRP but also including CPEB, pumilio, ZBP, caprin, Hu, Ago/miRNA complexes and the noncoding RNAs BC1 and BC200) through genetic and biochemical approaches has been quite successful, identification of the mRNAs regulated by these binding proteins remains a major hurdle. Previous approaches have included in vitro RNA selection [77, 78] and co-IP followed by either microarray (RIP-Chip, also called

Concluding remarks

Application of HITS-CLIP to other specific mRNA-binding proteins implicated in the control of activity-dependent translation in brain, such as CPEB, ZBP, Hu, caprin or pumilio should greatly expand our understanding of the mRNA targets they regulate. Indeed, application to Ago/miRNA complexes in P13 mouse brain has yielded a compelling map of in vivo Ago binding sites on mRNA [92]. Using HITS-CLIP analysis to quantify changes in binding owing to activity, or in subcellular fractions such as

References and recommended reading

Papers of particular interest published within the period of review have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I apologize to the many authors whose relevant work could not be cited owing to space limitations and thank Robert Darnell, Alicia Darnell and Sarah van Dreische for their critical review of the manuscript during its preparation, as well the past and present members of the Darnell laboratory for their insights. JCD is supported by NICHD grant R01 HD40647.

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