Local Dendritic Synthesis Research Articles

UPF2 leads to degradation of dendritically targeted mRNAs to regulate synaptic plasticity and cognitive function.

Synaptic plasticity requires a tight control of mRNA levels in dendrites. RNA translation and degradation pathways have been recently linked to neurodevelopmental and neuropsychiatric diseases, suggesting a role for RNA regulation in synaptic plasticity and cognition. While the local translation of specific mRNAs has been implicated in synaptic plasticity, the tightly controlled mechanisms that regulate local quantity of specific mRNAs remain poorly understood. Despite being the only RNA regulatory pathway that is associated with multiple mental illnesses, the nonsense-mediated mRNA decay (NMD) pathway presents an unexplored regulatory mechanism for synaptic function and plasticity. Here, we show that neuron-specific disruption of UPF2, an NMD component, in adulthood attenuates learning, memory, spine density, synaptic plasticity (L-LTP), and potentiates perseverative/repetitive behavior in mice. We report that the NMD pathway operates within dendrites to regulate Glutamate Receptor 1 (GLUR1) surface levels. Specifically, UPF2 modulates the internalization of GLUR1 and promotes its local synthesis in dendrites. We identified neuronal Prkag3 mRNA as a mechanistic substrate for NMD that contributes to the UPF2-mediated regulation of GLUR1 by limiting total GLUR1 levels. These data establish that UPF2 regulates synaptic plasticity, cognition, and local protein synthesis in dendrites, providing fundamental insight into the neuron-specific function of NMD within the brain.

Neurogranin Expression Is Regulated by Synaptic Activity and Promotes Synaptogenesis in Cultured Hippocampal Neurons.

Neurogranin (Ng) is a calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) and is highly enriched in the dendrites and spines of telencephalic neurons. It is proposed to be involved in regulating CaM availability in the post-synaptic environment to modulate the efficiency of excitatory synaptic transmission. There is a close relationship between Ng and cognitive performance; its expression peaks in the forebrain coinciding with maximum synaptogenic activity, and it is reduced in several conditions of impaired cognition. We studied the expression of Ng in cultured hippocampal neurons and found that both protein and mRNA levels were about 10% of that found in the adult hippocampus. Long-term blockade of NMDA receptors substantially decreased Ng expression. On the other hand, treatments that enhanced synaptic activity such as long-term bicuculline treatment or co-culture with glial cells or cholesterol increased Ng expression. Chemical long-term potentiation (cLTP) induced an initial drop of Ng, with a minimum after 15min followed by a slow recovery during the next 2-4h. This effect was most evident in the synaptosome-enriched fraction, thus suggesting local synthesis in dendrites. Lentiviral expression of Ng led to increased density of both excitatory and inhibitory synapses in the second and third weeks of culture. These results indicate that Ng expression is regulated by synaptic activity and that Ng promotes the synaptogenesis process. Given its relationship with cognitive function, we propose targeting of Ng expression as a promising strategy to prevent or alleviate the cognitive deficits associated with aging and neuropathological conditions.

Shifting patterns of polyribosome accumulation at synapses over the course of hippocampal long-term potentiation.

Hippocampal long-term potentiation (LTP) is a cellular memory mechanism. For LTP to endure, new protein synthesis is required immediately after induction and some of these proteins must be delivered to specific, presumably potentiated, synapses. Local synthesis in dendrites could rapidly provide new proteins to synapses, but the spatial distribution of translation following induction of LTP is not known. Here, we quantified polyribosomes, the sites of local protein synthesis, in CA1 stratum radiatum dendrites and spines from postnatal day 15 rats. Hippocampal slices were rapidly fixed at 5, 30, or 120 min after LTP induction by theta-burst stimulation (TBS). Dendrites were reconstructed through serial section electron microscopy from comparable regions near the TBS or control electrodes in the same slice, and in unstimulated hippocampus that was perfusion-fixed in vivo. At 5 min after induction of LTP, polyribosomes were elevated in dendritic shafts and spines, especially near spine bases and in spine heads. At 30 min, polyribosomes remained elevated only in spine bases. At 120 min, both spine bases and spine necks had elevated polyribosomes. Polyribosomes accumulated in spines with larger synapses at 5 and 30 min, but not at 120 min. Small spines, meanwhile, proliferated dramatically by 120 min, but these largely lacked polyribosomes. The number of ribosomes per polyribosome is variable and may reflect differences in translation regulation. In dendritic spines, but not shafts, there were fewer ribosomes per polyribosome in the slice conditions relative to in vivo, but this recovered transiently in the 5 min LTP condition. Overall, our data show that LTP induces a rapid, transient upregulation of large polyribosomes in larger spines, and a persistent upregulation of small polyribosomes in the bases and necks of small spines. This is consistent with local translation supporting enlargement of potentiated synapses within minutes of LTP induction.

Mechanism of the Dendritic Translation and Localization of Brain-derived Neurotrophic Factor.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3' UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3' UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

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 Ca(2+)- 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.

Distinct spatiotemporal expression of SAPAP transcripts in the developing rat brain: a novel dendritically localized mRNA

The four members of the family of synapse- associated protein 90/ postsynaptic density-95- associated proteins (SAPAP1–4) are adapter proteins of postsynaptic density (PSD). They interact with different synaptic scaffolding proteins, cytoskeletal components, and signalling components, and are therefore considered to assemble functional multiprotein units at synapses. Here, we analyzed the spatiotemporal expression of SAPAP1– SAPAP4 genes in postnatal rat brain by in situ hybridization. All four genes are expressed in many brain areas, leading to overlapping yet distinct mRNA distribution patterns. Moreover, two mRNAs encoding distinct SAPAP3 isoforms exhibit basically identical postnatal expression patterns. In the hippocampus, SAPAP1, SAPAP2, and SAPAP4 transcripts are restricted to cell body zones, whereas SAPAP3 mRNAs are also detected in molecular layers. Thus, SAPAP3 is one of the few PSD components whose local synthesis in dendrites may contribute to an input-specific adaptation of dendritic spine function.

Distinct spatiotemporal expression of SAPAP transcripts in the developing rat brain: a novel dendritically localized mRNA

The four members of the family of synapse-associated protein 90/postsynaptic density-95-associated proteins (SAPAP1–4) are adapter proteins of postsynaptic density (PSD). They interact with different synaptic scaffolding proteins, cytoskeletal components, and signalling components, and are therefore considered to assemble functional multiprotein units at synapses. Here, we analyzed the spatiotemporal expression of SAPAP1–SAPAP4 genes in postnatal rat brain by in situ hybridization. All four genes are expressed in many brain areas, leading to overlapping yet distinct mRNA distribution patterns. Moreover, two mRNAs encoding distinct SAPAP3 isoforms exhibit basically identical postnatal expression patterns. In the hippocampus, SAPAP1, SAPAP2, and SAPAP4 transcripts are restricted to cell body zones, whereas SAPAP3 mRNAs are also detected in molecular layers. Thus, SAPAP3 is one of the few PSD components whose local synthesis in dendrites may contribute to an input-specific adaptation of dendritic spine function.

Glycoprotein synthesis at the synapse: fractionation of polypeptides synthesized within isolated dendritic fragments by concanavalin A affinity chromatography

The synthesis of glycosylated proteins at postsynaptic sites was evaluated by combining metabolic labeling of isolated pinched-off dendritic fragments (synaptodendrosomes) with glycoprotein isolation by Con A affinity chromatography. Three major labeled proteins were detected (apparent molecular weights of 128, 42 and 19 kDa) along with seven minor polypeptides. Treatment of the glycoprotein fraction with N-glycosidase F led to shift in the apparent molecular weight of the bands. Also, label incorporation into glycoprotein species was blocked by tunicamycin. Thus, the three prominent polypeptides and most of the minor components of this fraction corresponded to bona fide N-glycoproteins. Incubation of synaptodendrosomes with cycloheximide also inhibited label incorporation into the isolated glycoproteins, indicating that the labeling resulted from local de novo synthesis. Subcellular fractionation revealed that the labeled glycoproteins were present in soluble and particulate fractions, mainly microsomes and synaptic membranes, and one of the species (42 kDa) appeared in the incubation medium, indicating secretion. In addition, these glycoproteins were dissimilarly distributed in several brain regions, and were expressed differentially during development, reaching their highest level of synthesis during the period of synaptogenesis. These results provide evidence for local dendritic synthesis of particular glycoprotein components of the synapse.