Stefan Kindler

Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities.

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD‐95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD‐95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C‐terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD‐95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD‐95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ‐binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD‐95).

Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs

In neurones, the somatodendritic microtubule‐associated protein 2 regulates the stability of the dendritic cytoskeleton. Its extrasomatic localization appears to be a multicausal mechanism that involves dendritic mRNA trafficking, a process that depends on a dendritic targeting element in the 3′ untranslated region. Two rat MAP2‐RNA trans‐acting proteins, MARTA1 and MARTA2, exhibit specific high‐affinity binding to the dendritic targeting element. We have now affinity‐purified MARTA1 from rat brain. Analysis of proteolytic peptides revealed that rat MARTA1 is the orthologue of the human RNA‐binding protein KSRP. Rat MARTA1 is a 74‐kDa protein that contains four putative RNA‐binding domains and is 98% identical to human KSRP. Both purified rat MARTA1 and human KSRP preferentially bind to the dendritic targeting element, but do not strongly interact with other investigated regions of mRNAs encoding microtubule‐associated protein 2 and α‐tubulin. In rat brain neurones and cultured neurones derived from superior cervical ganglia, MARTA1 is primarily intranuclear, but is also present in the somatodendritic cytoplasm. Thus, MARTA1 may play a role in nucleocytoplasmic mRNA targeting.

Characterization of Staufen 1 ribonucleoprotein complexes

In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

Synaptopodin, a molecule involved in the formation of the dendritic spine apparatus, is a dual actin/α‐actinin binding protein

Synaptopodin (SYNPO) is a cytoskeletal protein that is preferentially located in mature dendritic spines, where it accumulates in the spine neck and closely associates with the spine apparatus. Formation of the spine apparatus critically depends on SYNPO. To further determine its molecular action, we screened for cellular binding partners. Using the yeast two‐hybrid system and biochemical assays, SYNPO was found to associate with both F‐actin and α‐actinin. Ectopic expression of SYNPO in neuronal and non‐neuronal cells induced actin aggregates, thus confirming a cytoplasmic interaction with the actin cytoskeleton. Whereas F‐actin association is mediated by a central SYNPO motif, binding to α‐actinin requires the C‐terminal domain. Notably, the α‐actinin binding domain is also essential for dendritic targeting and postsynaptic accumulation of SYNPO in primary neurons. Taken together, our data suggest that dendritic spine accumulation of SYNPO critically depends on its interaction with postsynaptic α‐actinin and that SYNPO may regulate spine morphology, motility and function via its distinct modes of association with the actin cytoskeleton.

The Insulin Receptor Substrate of 53 kDa (IRSp53) Limits Hippocampal Synaptic Plasticity

IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.

Postsynaptic recruitment of Dendrin depends on both dendritic mRNA transport and synaptic anchoring.

Synaptic plasticity and memory formation involve remodeling of the postsynaptic cytoskeleton, a process that is in part based on both local translation of dendritic mRNAs and synaptic recruitment of newly synthesized proteins. The postsynaptic component Dendrin that is encoded by a dendritically localized mRNA is thought to modulate the structure of the synaptic cytoskeleton. However, molecular mechanisms that control extrasomatic Dendrin mRNA transport and postsynaptic protein recruitment are unknown. The data presented here reveal that Dendrin interacts with the cytoskeletal components α‐actinin and Maguk with inverted orientation (MAGI) or synaptic scaffolding molecule (S‐SCAM). The latter retains Dendrin in the cytoplasm of mammalian cells and prevents its nuclear import. Furthermore in neurons, postsynaptic clustering of Dendrin requires dendritic targeting of its messenger RNA (mRNA), a process that is mediated by a sequence motif within the 3′ untranslated region. In summary our finding suggest that postsynaptic recruitment of Dendrin appears to critically depend on both local protein synthesis and association with the synaptic scaffolding protein MAGI/S‐SCAM. Its nuclear localization capacity further points to a function in retrograde signaling from the synapse to the nucleus.

Shank1 mRNA: Dendritic Transport by Kinesin and Translational Control by the 5′Untranslated Region

Dendritic mRNA transport coupled with local regulation of translation enables neurons to selectively alter the protein composition of individual postsynaptic sites. We have analyzed dendritic localization of shank1 mRNAs; shank proteins (shank1–3) are scaffolding molecules of the postsynaptic density (PSD) of excitatory synapses, which are crucial for PSD assembly and the formation of dendritic spines. Live cell imaging demonstrates saltatory movements of shank1 mRNA containing granules along microtubules in both anterograde and retrograde directions. A population of brain messenger ribonucleoprotein particles (mRNPs) containing shank1 mRNAs associates with the cargo‐binding domain of the motor protein KIF5C. Through expression of dominant negative proteins, we show that dendritic targeting of shank1 mRNA granules involves KIF5C and the KIF5‐associated RNA‐binding protein staufen1. While transport of shank1 mRNAs follows principles previously outlined for other dendritic transcripts, shank1 mRNAs are distinguished by their translational regulation. Translation is strongly inhibited by a GC‐rich 5′untranslated region; in addition, internal ribosomal entry sites previously detected in other dendritic transcripts are absent in the shank1 mRNA. A concept emerges from our data in which dendritic transport of different mRNAs occurs collectively via a staufen1‐ and KIF5‐dependent pathway, whereas their local translation is controlled individually by unique cis‐acting elements.

Dendritic mRNA Targeting of Jacob and N-Methyl-d-aspartate-induced Nuclear Translocation after Calpain-mediated Proteolysis

Jacob is a recently identified plasticity-related protein that couples N-methyl-d-aspartate receptor activity to nuclear gene expression. An expression analysis by Northern blot and in situ hybridization shows that Jacob is almost exclusively present in brain, in particular in the cortex and the limbic system. Alternative splicing gives rise to multiple mRNA variants, all of which exhibit a prominent dendritic localization in the hippocampus. Functional analysis in primary hippocampal neurons revealed that a predominant cis-acting dendritic targeting element in the 3′-untranslated region of Jacob mRNAs is responsible for dendritic mRNA localization. In the mouse brain, Jacob transcripts are associated with both the fragile X mental retardation protein, a well described trans-acting factor regulating dendritic mRNA targeting and translation, and the kinesin family member 5C motor complex, which is known to mediate dendritic mRNA transport. Jacob is susceptible to rapid protein degradation in a Ca2+- and Calpain-dependent manner, and Calpain-mediated clipping of the myristoylated N terminus of Jacob is required for its nuclear translocation after N-methyl-d-aspartate receptor activation. Our data suggest that local synthesis in dendrites may be necessary to replenish dendritic Jacob pools after truncation of the N-terminal membrane anchor and concomitant translocation of Jacob to the nucleus.

Makorin Ring Zinc Finger Protein 1 (MKRN1), a Novel Poly(A)-binding Protein-interacting Protein, Stimulates Translation in Nerve Cells

Background: Synaptic activity induces translation of mRNAs in dendrites of neurons. Results: Makorin 1 (MKRN1) interacts with poly(A)-binding protein, stimulates translation, accumulates in neuronal dendrites after plasticity-inducing stimuli, and is associated with dendritic mRNAs. Conclusion: MKRN1 has the potential to locally control the translation of dendritic mRNAs at synapses. Significance: MKRN1 is a novel positive regulator of translation. The poly(A)-binding protein (PABP), a key component of different ribonucleoprotein complexes, plays a crucial role in the control of mRNA translation rates, stability, and subcellular targeting. In this study we identify RING zinc finger protein Makorin 1 (MKRN1), a bona fide RNA-binding protein, as a binding partner of PABP that interacts with PABP in an RNA-independent manner. In rat brain, a so far uncharacterized short MKRN1 isoform, MKRN1-short, predominates and is detected in forebrain nerve cells. In neuronal dendrites, MKRN1-short co-localizes with PABP in granule-like structures, which are morphological correlates of sites of mRNA metabolism. Moreover, in primary rat neurons MKRN1-short associates with dendritically localized mRNAs. When tethered to a reporter mRNA, MKRN1-short significantly enhances reporter protein synthesis. Furthermore, after induction of synaptic plasticity via electrical stimulation of the perforant path in vivo, MKRN1-short specifically accumulates in the activated dendritic lamina, the middle molecular layer of the hippocampal dentate gyrus. Collectively, these data indicate that in mammalian neurons MKRN1-short interacts with PABP to locally control the translation of dendritic mRNAs at synapses.