The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

AbstractMembers of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.

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The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014590 ◽

2020 ◽

Vol 295 (33) ◽

pp. 11776-11788

Author(s):

Molly Hodul ◽

Rakesh Ganji ◽

Caroline L. Dahlberg ◽

Malavika Raman ◽

Peter Juo

Keyword(s):

Mammalian Cells ◽

Deubiquitinating Enzymes ◽

Post Translational Modification ◽

Wd40 Repeat ◽

C Elegans ◽

Protein Levels ◽

Specific Protease ◽

The Stability ◽

And Function

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans. We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin–USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo. Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

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The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

10.1101/679415 ◽

2019 ◽

Author(s):

Molly Hodul ◽

Rakesh Ganji ◽

Caroline L Dahlberg ◽

Malavika Raman ◽

Peter Juo

Keyword(s):

Mammalian Cells ◽

Deubiquitinating Enzyme ◽

Deubiquitinating Enzymes ◽

Post Translational Modification ◽

Wd40 Repeat ◽

C Elegans ◽

Protein Levels ◽

The Stability ◽

And Function

ABSTRACTUbiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, mechanisms that regulate the deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from target proteins are poorly understood. We previously showed that the DUB USP-46 removes ubiquitin from the glutamate receptor GLR-1 and regulates it trafficking and degradation in C. elegans. We found that WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identify another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome promotes the abundance of USP-46, and this effect is non-additive with increased expression of WDR-48. We found that USP-46 is ubiquitinated, and expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the half-life of USP-46. A point mutant version of WDR-48 that disrupts binding to USP-46 is unable to promote USP-46 abundance in vivo. Together, these data support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism to control DUB availability and function.

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Tissue-specific regulation and function of Grb10 during growth and neuronal commitment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1411254111 ◽

2014 ◽

Vol 112 (22) ◽

pp. 6841-6847 ◽

Cited By ~ 30

Author(s):

Robert N. Plasschaert ◽

Marisa S. Bartolomei

Keyword(s):

Motor Neurons ◽

Cellular Proliferation ◽

Neuronal Development ◽

Embryonic Stem ◽

Specific Expression ◽

Tissue Specific ◽

Specific Regulation ◽

And Function

Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10’s unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.

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C. elegans germ granules require both assembly and localized regulators for mRNA repression

Nature Communications ◽

10.1038/s41467-021-21278-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Scott Takeo Aoki ◽

Tina R. Lynch ◽

Sarah L. Crittenden ◽

Craig A. Bingman ◽

Marvin Wickens ◽

...

Keyword(s):

Liquid Droplet ◽

Scaffolding Protein ◽

P Granules ◽

C Elegans ◽

Cytoplasmic Rna ◽

And Function ◽

Rnp Granules ◽

Key Residues ◽

Reporter Mrna

AbstractCytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

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DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

Biochemical Journal ◽

10.1042/bj20121085 ◽

2013 ◽

Vol 451 (3) ◽

pp. 453-461 ◽

Cited By ~ 26

Author(s):

Claudia C. S. Chini ◽

Carlos Escande ◽

Veronica Nin ◽

Eduardo N. Chini

Keyword(s):

Breast Cancer ◽

Nuclear Receptor ◽

Clock Genes ◽

Mrna Levels ◽

Protein Levels ◽

The Stability ◽

And Function ◽

Interfering Rna ◽

In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

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FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

10.21203/rs.3.rs-493867/v1 ◽

2021 ◽

Author(s):

Bin Qiu ◽

Zhaohui Zhong ◽

Shawn Righter ◽

Yuxue Xu ◽

Jun Wang ◽

...

Keyword(s):

Hippocampal Neurons ◽

Translational Regulation ◽

Disease Onset ◽

Fold Increase ◽

Knock Out ◽

Fk506 Binding Protein ◽

Protein Levels ◽

And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

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Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity

10.1101/2021.03.15.435499 ◽

2021 ◽

Author(s):

Stephen M Blazie ◽

Seika Takayanagi-Kiya ◽

Katherine A McCulloch ◽

Yishi Jin

Keyword(s):

Rna Binding ◽

Mrna Translation ◽

Initiation Factor ◽

Translation Efficiency ◽

Dependent Manner ◽

Control Activity ◽

C Elegans ◽

Protein Levels ◽

Neuronal Protein

AbstractThe translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of theC. elegansRNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR dependent manner. Our study reveals anin vivomechanism for eIF3 in governing neuronal protein levels to control activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.

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rab-27 acts in an intestinal pathway to inhibit axon regeneration in C. elegans

PLoS Genetics ◽

10.1371/journal.pgen.1009877 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1009877

Author(s):

Alexander T. Lin-Moore ◽

Motunrayo J. Oyeyemi ◽

Marc Hammarlund

Keyword(s):

Motor Neurons ◽

Axon Regeneration ◽

Rab Gtpase ◽

Long Distance ◽

Extrinsic Factors ◽

C Elegans ◽

Cellular Environment ◽

Neuropeptide Secretion ◽

Neuronal Tissues ◽

Nervous System Function

Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.

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In Vivo Ablation of the Conserved GATA-Binding Motif in the Amh Promoter Impairs Amh Expression in the Male Mouse

Endocrinology ◽

10.1210/en.2019-00047 ◽

2019 ◽

Vol 160 (4) ◽

pp. 817-826 ◽

Cited By ~ 6

Author(s):

Marie France Bouchard ◽

Francis Bergeron ◽

Jasmine Grenier Delaney ◽

Louis-Mathieu Harvey ◽

Robert S Viger

Keyword(s):

Developmental Time ◽

Gonadal Development ◽

Conclusive Evidence ◽

Binding Motif ◽

Male Embryo ◽

Steroidogenic Factor 1 ◽

Protein Levels ◽

Positive Modulator ◽

And Function

Abstract GATA4 is an essential transcriptional regulator required for gonadal development, differentiation, and function. In the developing testis, proposed GATA4-regulated genes include steroidogenic factor 1 (Nr5a1), SRY-related HMG box 9 (Sox9), and anti-Müllerian hormone (Amh). Although some of these genes have been validated as genuine GATA4 targets, it remains unclear whether GATA4 is a direct regulator of endogenous Amh transcription. We used a CRISPR/Cas9-based approach to specifically inactivate or delete the sole GATA-binding motif of the proximal mouse Amh promoter. AMH mRNA and protein levels were assessed at developmental time points corresponding to elevated AMH levels: fetal and neonate testes in males and adult ovaries in females. In males, loss of GATA binding to the Amh promoter significantly reduced Amh expression. Although the loss of GATA binding did not block the initiation of Amh transcription, AMH mRNA and protein levels failed to upregulate in the developing fetal and neonate testis. Interestingly, adult male mice presented no anatomical anomalies and had no evidence of retained Müllerian duct structures, suggesting that AMH levels, although markedly reduced, were sufficient to masculinize the male embryo. In contrast to males, GATA binding to the Amh promoter was dispensable for Amh expression in the adult ovary. These results provide conclusive evidence that in males, GATA4 is a positive modulator of Amh expression that works in concert with other key transcription factors to ensure that the Amh gene is sufficiently expressed in a correct spatiotemporal manner during fetal and prepubertal testis development.

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Microtubule-dependent ribosome localization in C. elegans neurons

eLife ◽

10.7554/elife.26376 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Kentaro Noma ◽

Alexandr Goncharov ◽

Mark H Ellisman ◽

Yishi Jin

Keyword(s):

Cell Biology ◽

Neuronal Development ◽

Ultrastructural Level ◽

Synthesis Methods ◽

Tissue Specific ◽

C Elegans ◽

Multicellular Organisms ◽

Axonal Trafficking ◽

Insight Into

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

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