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you may want to ask where is the SITE of protein synthesis
The small GTPase RAB10 also regulates tubular ER formation. However, it is involved in ER tubule growth independently of ATL1 (ref. ). To examine whether RAB10 also influences protein synthesis and dendritic spine formation, the effects of WT and a GDP-locked T23N mutant of RAB10 in cultured hippocampal neurons were compared. The RAB10 T23N mutant had been shown to inhibit the function of endogenous RAB10 in tubular ER formation. As expected, inhibition of endogenous RAB10 activity by expression of the RAB10 T23N mutant impaired the ER distribution in neurons (). Although the mechanisms underlying the function of ATL1 and RAB10 in ER formation differ, we found that similar to ATL1, the RAB10 T23N mutant also reduced protein synthesis and dendritic spine formation (). Together, the ATL1 and RAB10 results echo our hypothesis that regulation of the tubular ER network controls protein synthesis and consequently influences the density of dendritic spines.
Down syndrome critical region1 (DSCR1, also called RCAN1 or regulator of calcineurin) is located on human chromosome 21 and highly expressed in the brain, especially enriched in hippocampal neurons (). DSCR1 belongs to a conserved family of calcineurin (CaN) inhibitors called calcipressins, which includes RCN1P in yeast (), CBP1 in fungus (), nebula in Drosophila (), as well as DSCR1 in mouse and human (; ). DSCR1 knockout mice show learning deficits and impaired late-phase long-term potentiation (L-LTP), which requires new gene expression (). By blocking translation and transcription surgically or using inhibitors, several reports have shown that L-LTP requires local protein synthesis, while somatic transcription and translation may be dispensable (; ). Together, it implies that DSCR1 is involved in local protein synthesis. Furthermore, a transgenic mouse overexpressing DSCR1 in the brain show significant defects in learning (), suggesting that DSCR1 may play an important role in intellectual disability in Down syndrome. However, the roles of DSCR1 in dendritic spine morphogenesis or local protein synthesis at dendritic spines are not studied.
Protein Synthesis -Translation and Regulation
According to our hypothesis, BDNF treatment triggers phosphorylation of DSCR1, which may activate calcineurin and lead to increased dephosphorylation of phospho-FMRP. To test this model, we determined if DSCR1 could be phosphorylated by endogenous kinase(s) found in the mouse brain. This was achieved by incubating immunoprecipitated Flag-DSCR1 from HEK cells with extracts of mouse brain or hippocampal neurons treated with or without BDNF. The immunoprecipitated Flag-DSCR1 from HEK cells is not phosphorylated, however, showed that Flag-DSCR1 becomes phosphorylated after incubation with brain extracts or BDNF-treated hippocampal neuronal extracts, as revealed by western blotting using phosphorylated serine-specific antibody. We then determined whether phosphorylated DSCR1 would cause active calcineurin. Calcineurin activity assays revealed that Flag-DSCR1 inhibits calcineurin activity, but Flag-DSCR1 treated with brain extracts before the assay showed higher calcineurin activity (). Also, Flag-DSCR1 incubated with extracts of hippocampal primary neurons stimulated with BDNF showed more calcineurin activity compared to that of neurons without BDNF treatment (). Taken together, these data show that BDNF stimulation increases DSCR1 phosphorylation, which results in active calcineurin, thus leading to increased dephosphorylation of phospho-FMRP.
We next investigated the underlying molecular basis of how DSCR1 regulates local protein synthesis. suggested that non-phosphorylated Rcn1, a yeast homologue of DSCR1, inhibits calcineurin, while phosphorylation of Rcn1 by GSK-3 may activate calcineurin. It is plausible that BDNF stimulation on dendritic spines triggers phosphorylation of DSCR1, thus DSCR1 no longer functions as an inhibitor of calcineurin. Subsequently, active calcineurin dephosphorylates phospho-FMRP, and in turn FMRP dissociates from target mRNAs thus allowing local mRNA translation to occur. To test this model, we first determined if BDNF treatment of hippocampal neurons alters FMRP phosphorylation. After BDNF treatment, the intensity of phospho-FMRP reduced significantly while the level of DSCR1 and the amount of FMRP remain mostly unaltered, suggesting that phospho-FMRP is dephosphorylated following BDNF treatment (; ). Next, we tested if BDNF treatment of hippocampal neurons elevates calcineurin activity. As shown in , calcineurin activity is indeed increased in BDNF-treated hippocampal neurons. These results suggest that calcineurin may act on phospho-FMRP after BDNF stimulation. To verify if calcineurin can dephosphorylate phospho-FMRP, we immunoprecipitated phospho-FMRP from HEK cells with phospho-FMRP-specific antibody, then added purified calcineurin to the immunoprecipitates. Calcineurin indeed dephosphorylated phospho-FMRP (). In addition, we examined the level of phospho-FMRP in control and DSCR1−/− transgenic mice. We found that the level of pFMRP is decreased in DSCR1−/− mice (), suggesting that the ability of DSCR1 to regulate calcineurin leads to modulation of pFMRP. We also treated primary hippocampal neurons with BDNF in the presence of calcineurin inhibitor, cyclosporin A, and measured the level of phospho-FMRP. We found that in the presence of calcineurin inhibitor, BDNF no longer triggered dephosphorylation of pFMRP, confirming that BDNF induced dephosphorylation of phospho-FMRP acts through calcineurin.
Only sponges and a few other simpler animals lack neurons
In addition to BONCAT, we applied another method—surface sensing of translation (SUnSET)—to monitor protein synthesis through the incorporation of puromycin into neurons. Puromycin resembles the 3′ end of transfer RNA and incorporates into synthesizing polypeptide chains. Puromycin-labeled proteins can then be detected using puromycin antibody. Similar to the results using AHA labeling, Vcp knockdown reduced puromycin labeling and P47 overexpression rescued puromycin incorporation in Vcp knockdown neurons (). In conclusion, both BONCAT and SUnSET reveal the protein synthesis defects caused by Vcp and P47 deficiencies.
To more closely examine its control over dendritic spine density, in addition to its effect on protein synthesis revealed by AHA and puromycin labeling, we also wondered whether Vcp deficiency influences the expression of synaptic membrane proteins. We screened several glutamate receptors, which are enriched at dendritic spines and are critical for their functioning, and found that expression levels of GRIN2B and GRIA2/3 were noticeably lower in VCP R95G knockin neurons (). GRIN2A levels also tended to be lower in VCP R95G knockin neurons, but the difference did not attain statistical significance. For GRIA1 and GRM5, their protein levels were comparable between WT and VCP R95G knockin neurons (). PSD-95 protein levels were also unaffected by the VCP R95G mutation (). These results suggest that some synaptic ion channels are particularly sensitive to the VCP R95G mutation.
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Peptide Hormones - The Medical Biochemistry Page
In our experiments, we did find an unfolded protein response induced by Ufd1l knockdown and tunicamycin treatment. However, the dendritic spine density was not affected by Ufd1l knockdown or tunicamycin treatment, suggesting that an unfolded protein response is not involved in regulation of dendritic spine density. It is noteworthy that protein translation was transiently suppressed by tunicamycin treatment in neurons. Perhaps protein translation suppression is too transient to noticeably influence the total amounts of synaptic membrane proteins, thereby explaining the lack of effect on dendritic spines. Although an unfolded protein response, either induced by Ufd1l knockdown or tunicamycin treatment, seems not to be critical for regulation of dendritic spine density under our experimental conditions, it is still possible that an unfolded protein response influences other neuronal functions. This would be a topic worth pursuing in the future. In addition to protein degradation, the VCP–UFD1L–NPL4 complex also regulates other activities, such as the ubiquitin-governed DNA damage response. As DNA double-stranded breaks are involved in expression of neuronal early-response genes, it would also be intriguing to investigate whether VCP contributes to regulation of neuronal early response genes. It may not directly control dendritic spine formation but likely regulates other neuronal functions.
FSP Syndromes - Washington University in St. Louis
In this report, we unexpectedly found that the ER is the critical target of Vcp deficiency in regulating dendritic spine density in neurons. Our data suggest that VCP, P47 and ATL1 work together to control tubular ER formation and influence protein synthesis. Previous studies have suggested that the VCP/P47 complex regulates the activity of an unknown membrane fusion regulator, thereby controlling tubular ER formation. Based on our data, it seems possible that ATL1 is the membrane fusion regulator of the VCP-P47 complex that regulates ER membrane fusion. In our TEM analysis, we found that the length of rER, ribosomal attachment on rER and rER abundance were noticeably reduced in VCP R95G knockin neurons. Although rER is generally believed to contribute to synthesis of membrane/secreted proteins, several studies have demonstrated that a noticeable fraction of cytosolic proteins are also synthesized by ribosomes associated with ER,,. Thus, ER defects caused by Vcp deficiency likely results in a protein synthesis deficit. Using 1-h AHA labeling, we show here that protein synthesis efficiency is impaired by a Vcp deficiency, which is consistent with the ER defects revealed by TEM. Further, expression of synaptic glutamate receptors GRIN2B and GRIA2/3—the important ion channels of excitatory synapses—was reduced in VCP R95G knockin mutant neurons, indicative of VCP involvement in the impairment of the synaptic response and consequent elimination of dendritic spines. It would be intriguing to explore in the future whether the VCP R95G mutation results in impairment of electrophysiological responses and cognitive deficiencies using our knockin mutant mice.
ER stress and the unfolded protein response - …
In this report, we provide evidence that changes in ER structure and function may be a common characteristic of various neurological disorders, including IBMPFD and SPG3A. SPG3A is characterized by progressive motor weakness and spasticity. Degeneration of corticospinal tract axons with the onset of childhood is the major phenotypic feature of SPG3A. In addition, recent studies have also indicated hypometabolism in the frontal cortex and cerebellum and functional disorders in the frontal cortex in patients with SPG3A,. Our data indicate that expression of the ATL1 mutant reduces the efficiencies of protein synthesis and dendritic spine formation in cultured hippocampal neurons. It seems likely that protein synthesis is also involved in the pathogenesis of SPG3A.
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