Cullin-RING E3 ubiquitin ligase 4 controls axonal morphogenesis during neuronal development

doi:10.21203/rs.3.rs-1972814/v1

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Cullin-RING E3 ubiquitin ligase 4 controls axonal morphogenesis during neuronal development

https://doi.org/10.21203/rs.3.rs-1972814/v1

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Neuritogenesis is critical for the establishment of proper neuronal connections during brain development. Thus, its failure causes neurodevelopmental defects such as intellectual disabilities. Cullin-RING E3 ubiquitin-ligase complexes are involved in neurodevelopmental processes including neurite outgrowth, migration, and differentiation via regulation of protein stability. In this study, we demonstrate a novel regulatory function of Cullin-RING E3 ubiquitin-ligase 4 (CRL4) in neurite morphogenesis during early neurodevelopment. Cul4a and Cul4b, core scaffold proteins of CRL4, are highly expressed and activated in the cytosolic compartment of developing neuron, and they are regulated by neuronal stimulation via N-methyl D-aspartate (NMDA) receptor signaling. CRL4 also interacts with cytoskeleton-regulating proteins involved in neurite morphogenesis in neurons. Notably, CRL4 inhibition enhances axonal extension and branching in developing neurons. Conversely, Cul4a overexpression suppresses basal and NMDA-enhanced axonal outgrowth. Furthermore, CRL4 regulates the stability of Doublecortin protein recruited by Cereblon. Taken together, we suggest a novel role of CRL4 in proper axonal morphogenesis in developing neurons by regulating cytoskeleton-regulating proteins.

neuritogenesis

CRL4

axon

cullin

Neurons are connected by forming synapses in a complicated but orderly manner, thereby establishing neural circuits for high-order brain function¹. Developing neurons migrate to proper locations, mature with polarized structures including axons and dendrites, and finally create synapses. Throughout these neurodevelopmental processes, neuronal morphogenesis, especially neuritogenesis, is critical developmental event². Sphere-shaped neural precursor cells undergo initial budding by the asymmetric and focal accumulation of cortical actin³. The sprouted buds develop into extended actin-rich filopodia and lamellipodia before becoming microtubule-stabilized neurites classified as axons or dendrites. These neurites massively extend and branch to form complex synaptic connections. Therefore, neuritogenesis requires spatiotemporally dynamic changes in cytoskeletal structures that mainly mediate cellular morphological changes⁴.

Actin-filament and microtubule, the main filamentous structures of neurite, are largely regulated by polar polymerization and depolymerization of globular actin and heterodimeric α/β-tubulin, respectively⁴. These processes are also controlled by GTP/GDP-exchanging regulators and stabilizing/destabilizing enzymes, including Profilin/Cofilin and Tau/Stathmin. In addition, cross-linking proteins of actin-filament and microtubule such as Doublecortin (Dcx) and Drebrin (Dbn1), and protein-trafficking motor proteins including Myosin and Kinesin/Dynein, are also involved in the coordination of actin-filaments and microtubules³. Thus, genetic mutations in cytoskeletal components or cytoskeleton-regulating proteins are highly implicated in neurodevelopmental diseases such as intellectual disability and autism^2,5. Therefore, cytoskeletal protein regulation is critical for proper neuritogenesis.

The ubiquitination-proteasome system has been proposed as a negative regulator of neuritogenesis⁶. Previous studies demonstrated that Smurf1, an E3 ubiquitin ligase, promotes neuritogenesis by ubiquitination-mediated degradation of RhoA, which regulates actin dynamics⁷. Recently, Cullin-RING E3 ubiquitin ligases (CRLs), the most prominent E3 ubiquitin ligase family in eukaryotes, have been implicated in neuritogenesis. They are multi-protein complexes formed by the core scaffold protein cullin, which serves as a platform for the E2 enzyme and substrates with the RING-finger protein Rbx1/2^8,9. Genetic depletion of the Cul3 in CRL3 induces abnormal accumulation of cytoskeletal proteins involved in neuronal migration and dendritogenesis, thereby leading to social and cognitive impairments implicated with autism spectrum disorder^10,11. Similarly, Kelch-like 15 (Klhl15), a substrate adaptor of CRL3 implicated in X-linked intellectual disability, promotes destabilization of the Dcx protein, thereby inhibiting dendrite outgrowth¹². Meanwhile, genetic mutations in CUL4B, a scaffold protein of CRL4, are linked to X-linked intellectual disability^13,14. Cul4b knockout mice exhibit abnormal dendritic morphology in the interneurons of the hippocampus, resulting in spatial learning defects and epileptic hypersensitivity¹⁵. However, the mechanism underlying the role of CRL4 in neuritogenesis remains unknown.

In this study, we explore how CRL4 is regulated, and how it is involved in neurological function during neurodevelopment. In conclusion, we propose that CRL4 is a novel regulatory component in neuritogenesis during early neuronal development.

Experimental animals

All animals were handled according to Laboratory Animal Resource Center and Daegu Gyeongbuk Institute of Science and Technology IACUC guidelines (Approval No. DGIST-IACUC-20102701-0000). All experimental protocols were approved by the Institutional Animal Care and Use Committees of DGIST. Sprague-Dawley rats were obtained from KOATECH (Korea).

Rat primary cerebral neuron culture and PC12 cell culture

On rat embryonic day 18, the cerebral cortex was dissected in pre-chilled Hank’s buffered salt solution (#14025, Gibco, Waltham, MA, USA), which is supplemented with 6% glucose (Sigma-Aldrich, St. Louis, MO, USA) and 50 unit per ml penicillin-streptomycin (#15140, Gibco, Waltham, MA, USA). The cerebral cortices were trypsinized and physically dissociated by pipetting into single neurons. The dissociated neurons were plated at a density of 1*10⁵ cells per cm² onto glass coverslips, plates, or dishes which is pre-coated with poly-D-lysine (#P6407, Sigma-Aldrich, St. Louis, MO, USA) and fed in neurobasal media (#21103, Gibco, Waltham, MA, USA) containing 2% B27 supplement (#17504, Gibco, Waltham, MA, USA), 0.5 mM L-glutamine (#25030, Gibco, Waltham, MA, USA) and 50 unit per ml penicillin/streptomycin. The neurons were incubated in 5% CO₂ at 37℃. And then, half of the medium was exchanged with fresh media per three days.

PC12 cell line (#CRL-1721™) was purchased from ATCC (Manassas, VA, USA). PC12 cells were seeded on a 12-well or 6-well plate coated by poly D-lysine (0.01%; Sigma Chemical Co., USA) and grown in RPMI 1640 medium (#11875093, Gibco, USA) containing 10% horse serum (#26050088, Gibco, USA), 5% fetal bovine serum (FBS) (#SH30919.03, Hyclone, USA), and 1% penicillin/streptomycin at 37°C with 5% CO₂.

Chemicals

A set (#ab120376) of glutamate receptor antagonists (D-AP5, NBQX disodium salt, and LY341495 sodium salt), Memantine (#ab146687), MLN4924 (#ab216470) and NMDA (#ab120052) were commercially purchased from Abcam (Cambridge, MA, USA).

Immunoblotting and Antibodies

Cultured cerebral neurons were collected in lysis buffer (125mM Tris-Cl, 4% SDS, pH6.8) with a protease and phosphatase inhibitor cocktail (#78442, ThermoFisher Scientific, Waltham, MA, USA). The protein concentration of the lysates was measured using a BCA assay (#23225, ThermoFisher Scientific, Waltham, MA, USA). And then, the lysates were loaded into SDS-PAGE and transferred into PVDF membrane (#IPVH00010, Millipore, Darmstadt, Germany). The membranes were blocked with 5% non-fat milk (#232100, BD, Franklin Lakes, NJ, USA) in TBST at room temperature for 1 hr and treated by primary antibodies for over 2 hr. After washing with TBST, HRP-conjugated antibodies were treated, washed with TBST, and immunoblotted using Pierce ECL western blotting substrate (#23225, ThermoFisher Scientific, Waltham, MA, USA). Used antibodies were anti-Cul4a (#ab72548, Abcam, Cambridge, MA, United States), anti-Cul4b (#66038-1-lg, Proteintech, Rosemont, IL, USA), anti-Ddb1 (#ab9194, Abcam, Cambridge, MA, USA), anti-Actb (#MAB1501R, Millipore, Darmstadt, Germany), anti-Lmnb1 (#ab16048, Abcam, Cambridge, MA, USA), anti-Dcx (#sc-271390, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Myh10 (#MA5-27767, Invitrogen, Waltham, MA, USA), anti-acetylated α-tubulin (#sc-23950, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Crmp2 (#sc-376739, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mCherry (#ab125096, Abcam, Cambridge, MA, USA), and anti-Flag (#F1804, Sigma-Aldrich, St. Louis, MO, USA)

Immunocytochemistry and proximity ligation assay

Cultured neurons were rinsed with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. After washing out with PBST (0.03% Triton X-100 in PBS), cells were incubated with a blocking solution (3% BSA in PBST) at room temperature for 30 min. The primary antibodies, diluted into a blocking solution with proper titer (1:100–200), were treated at room temperature for 2 hr. Used antibodies were anti-Cul4a, anti-Cul4b, anti-Ddb1, anti-Tau (#ab75714, Abcam, Cambridge, MA, USA), anti-Crbn (#PA5-61122, Invitrogen, Waltham, MA, USA), anti-Myc (#sc-40, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HA (#sc-7392, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-Flag. After washing with PBST, fluorescence-conjugated secondary antibodies were diluted in a blocking solution (1:200) and treated for 1 hr. The specimen was mounted onto slide grass with a DAPI-contained mounting solution (#H-1200, Vectashield, Burlingame, CA, USA). For proximity ligation assay ¹⁶, used Duolink® In Situ Orange Starter Kit Mouse/Rabbit (#DUO92102, Sigma-Aldrich, St. Louis, MO, USA) and Goat/Rabbit (#DUO92106, Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s protocol.

Immunohistochemistry

The Sprague-Dawley rats were anesthetized by using Zoletil 50 (Virbac, Carros, France) (50mg/kg) and Rompun (Bayer AG, Leverkusen, Germany) (5 ~ 10mg/kg), transcardially perfused with cold saline (0.9% NaCl), and fixed with pre-chilled 4% paraformaldehyde in PBS. Brain tissues were dissected, dehydrated by 2-hr serial incubations with 70%, 85%, 95%, and 100% ethanol (#1.00983.1011, Merck, Darmstadt, Germany), soaked in xylene (#117, Duksan, Seoul, South Korea) for 4 hr, and finally embedded into paraffin for 4 hr. The paraffin-embedded samples were sectioned at five µm thickness by a rotary microtome (Leica, Buffalo Grove, IL, United States). The sections were attached to the micro slide glass (#BM5516, Muto Pure Chemicals, Tokyo, Japan). For antigen retrieval, they were treated with 0.01 M citric acid monohydrate (pH 6.0) at 100℃ for 5 min and permeated in 3% hydrogen peroxide for 30 min. And then, they were incubated with blocking solution (5% normal donkey serum in PBST) at room temperature for 1 hr and treated with primary antibodies, diluted in blocking solution, at 4℃ for over 16 hr. After several washes, secondary fluorescence-conjugated antibodies were treated for 1 hr at room temperature and mounted with a DAPI-contained mounting solution.

Fractionation of nucleus and cytosol

Neurons were collected in pre-chilled fractionation buffer (250 mM sucrose, 10 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES, protease and phosphatase inhibitor cocktails, pH7.5). The lysate was passed through a 27-gauge needle with a syringe 10-times and centrifuged at 500 ×g for 15 min at 4°C. The pellet containing the nucleus-enriched fraction and the supernatant containing the cytosolic fraction were separately collected. The pellet was resuspended into a fractionation buffer for nuclear-enriched fraction, passed through a 27-gauge needle, and centrifuged at 500 ×g for 15 min. And this process was repeated twice to clear residual other cellular compartments. The final pellet was lysed in lysis buffer (125 mM Tris-Cl, 4% SDS, pH6.8). For cytosolic fraction, the supernatant was centrifuged at 10000 ×g for 15 min at 4°C. The residual pellet, which contains a membrane-enriched fraction, was discarded, and the supernatant was resuspended into fractionation buffer and centrifuged at 10000 ×g for 15 min to wash other cellular compartments. This process was performed twice, and the final supernatant was lysed in lysis buffer.

Immunoprecipitation

Cultured neurons were lysed with immunoprecipitation buffer (100 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl, and 0.5% Triton X-100, pH7.4) and passed through a 28-gauge needle with a syringe ten times to further lyse. The lysate was centrifuged at 13200 rpm at 4℃ for 20 min, and its supernatant was collected. The lysate was pre-cleared with protein-A/G agarose beads (#20421, ThermoFisher Scientific, Waltham, MA, USA), and its protein amount was measured by BCA assay (#23225, ThermoFisher Scientific, Waltham, MA, USA). Primary antibodies were treated in the lysate with slow rotation at 4℃ overnight. The next day, the protein-A/G agarose bead was incubated with the sample for 2 hr, spun down at 2000 ×g for 3 min, and washed with immunoprecipitation buffer. The immunoreacting protein-A/G agarose bead was lysed with SDS loading buffer at 95℃ for 10 min. And then, its supernatant was loaded with SDS-PAGE, and further experiments were performed.

LC-MS/MS

Nano LC-MS/MS analysis was performed with a nano HPLC system (Agilent, Wilmington, DE). The nanochip column (Agilent, Wilmington, DE, 43 mm × 0.075 mm) was used for peptide separation. Mobile phase A for LC separation was 0.1% formic acid in deionized water, and mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 3% B to 45% B in 30 min, 45% B to 95% B in 1 min, 95% B in 4 min, and 3% B in 10 min. The flow rate was maintained at 300 nl/min. Product ion spectra were collected in the information-dependent acquisition (IDA) mode and were analyzed by Agilent 6530 Accurate-Mass Q-TOF using continuous cycles of one full scan TOF MS from 300–2000 m/z (1.0 s) plus three product ion scans from 150–2000 m/z (1.5 s each). Precursor m/z values were selected starting with the most intense ion, using a selection quadrupole resolution of 3 Da. The rolling collision energy feature was used, which determines collision energy based on the precursor value and charge state. The dynamic exclusion time for precursor ion m/z values was 60 s.

The mascot algorithm (Matrixscience, USA) was used to identify peptide sequences in a protein sequence database. Database search criteria were taxonomy; Rattus (148853 sequences; 70437683 residues), fixed modification; carbamidomethylated at cysteine residues, variable modification; oxidized at methionine residues, maximum allowed missed cleavage; 2, MS tolerance; 100 ppm, MS/MS tolerance; 0.1 Da. Only peptides resulting from trypsin digests were considered. The peptides were filtered with a significance threshold of p < 0.05.

DNA Constructs and gene transfection

pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909). And pcDNA3-DN-hCUL4A-FLAG and pcDNA3-DN-hCUL4B-FLAG were gifts from Wade Harper (Addgene plasmid #15821, #15822). pHIV-EGFP was a gift from Bryan Welm & Zena Werb (Addgene plasmid #21373). For DN-Cul4a and DN-Cul4b, we obtained blunt-ended PCR products from pcDNA3-DN-hCUL4A-FLAG or pcDNA3-DN-hCUL4B-FLAG and then inserted them into HpaI-digested pHIV-EGFP. Myc-tagged rat Cul4b-1, -x1, and -x2 isoforms were generated by inserting each PCR product into BamHI and XhoI of pcDNA3. The PCR products were obtained from the cDNA of rat cultured cerebral neurons using DNA oligomers. For Cul4b-1, 5’-CAG GAT CCC TTC ACG TTC AAC TAA GTC TAG-3’; for Cul4b-x1, 5’-CAG GAT CCC TTG GCG CGA AGC TCA CCG CGG-3’; for Cul4b-x2, 5’-CAG GAT CCC TGC TGA GGA ATC TTC CTC CTC-3’; for reverse primer of all three Cul4b isoforms, 5’-TGC TCG AGC TAT GCA ATA TAG TTG TAC T-3’ were used. For pcDNA-V5-tagged Crbn, the same cDNA of rat cultured cerebral neuron was also used. The PCR product was obtained with DNA oligomers; 5’- GCC TCG AGA TGG CCG GCG AGG GAG ATC-3’ and 5’- GCC CGC GGT AAG CAA AGT ATT ACT TTG-3’ was inserted into pcDNA3.2-DEST vector after digested with SacII and Xhol.

We used CalPhos Mammalian Transfection Kit (#631312, Clontech, Japan) for gene transfection into cultured neurons, according to the manufacturer’s protocol. And lipofectamine 3000 (#L3000001, Thermo Fisher Scientific, Waltham, MA, USA) was used for gene transfection into PC12 cells, according to the manufacturer’s protocol.

Quantification of mRNA level

We extracted total RNA from cultured neurons using RNeasy Plus Mini Kit (#74136, Qiagen, Düssldorf, Germany) and followed the manufacturer’s protocol. The mRNA was reverse-transcripted to cDNA using SuperScript IV Reverse Transcriptase (#18090010, Thermo Fisher Scientific, Waltham, MA, USA). We performed PCR using Herculase II Fusion DNA Polymerase (#600677, Agilent, Santa Clara, CA, USA) with these primers. For Cul4a, 5’-TGA GTT GCG AAG AAC ACT GC-3’ and 5’-CAC GCC ACG TAG TGG TAC TG-3’; for Cul4b, 5’-CTT TTT CAA ACC CTG GTG CT-3’ and 5’-GTC CCG GTC AAT TAG GGA TT-3’; for Cul4b-1, 5’-GGC AAA AAG AGG ATG TCA CG-3’; for Cul4b-x1, 5’-TAA GGA ACG GGT GGC TGA T-3’; for Cul4b-x2, 5’-TTA CCT GTC TGC CTC CTT CG-3’; for reverse primer of all three Cul4b isoforms, 5’-AGA TTC CTC AGC CAT TTT CG-3’; for Ppia, 5’-AGC ACT GGG GAG AAA GGA TT-3’ and 5’-AGC CAC TCA GTC TTG GCA GT-3’ were used.

Quantification of neurite morphology

To quantify neurite morphology, we first fixed the cultured neurons expressing GFP with 4% paraformaldehyde in PBS, then enhanced the GFP signal by staining with anti-GFP. We obtained neurons’ images using a confocal inverted or upright laser-scanning microscope (LSM700 or LSM780, Carl Zeiss, Germany). Each neuronal morphology was semi-automatically traced and skeletonized by using the Simple Neurite Tracer (SNT) plugin of the FIJI program (NIH, USA)¹⁷. The SNT plugin calculated the number and length of total neurites and branches based on the neurite traces. 50 cells were measured in each condition to analyze neurite morphology.

Statistical analysis

Statistic information is described in the figure legends. All experiments were performed at least three times from independent cell or animal preparations. Repeats for experiments or cell numbers are given in the figure legends as ‘n.’ Error bars represent ± standard error (SEM). Statistical analyses and graph plotting were conducted by using the Sigmaplot12.0 program. Throughout the study, p < 0.05 was considered significant.

Cul4a and Cul4b are up-regulated during early neuronal development

Unlike other CRLs, CRL4 is differentially formed by two paralogue cullin proteins, Cul4a and Cul4b, having over 80% sequence identity together with an adaptor protein, damage-specific DNA-binding protein 1 (Ddb1), and a substrate-recognizing Ddb1/Cul4-associated factor (DCAF)⁸. Thus, we examined the differential expression Cul4a and Cul4b at the developmental stage of cultured cerebral neurons. Immunoblot results showed that the levels of Cul4b and Ddb1 proteins dramatically decreased from in vitro day 5 (DIV5) to DIV10, while Cul4a protein was steadily expressed until DIV17 (Fig. 1a,b). Similarly, the level of Cul4b mRNA dramatically decreased from DIV5 to DIV10, while Cul4a mRNA slightly increased (Supplementary Fig. S1). These results indicate that Cul4b is transcriptionally regulated during neuronal development. Notably, Cul4a and Cul4b showed higher molecular weight shifts of approximately 10 kDa in immunoblots (Fig. 1a). Cullin proteins undergo neddylation, a covalent conjugation with neural-precursor-cell-expressed developmentally downregulated 8 (Nedd8), and it promotes CRL activation¹⁸. Therefore, we examined whether the molecular weight shifts in Cul4a and Cul4b were caused by neddylation. Treatment with MLN4924, an inhibitor of Nedd8-activating enzyme E1 (Nae1), dramatically abolished the band shifts of Cul4a and Cul4b on immunoblot, whereas treatment with CSN5i-3, an inhibitor of the Nedd8 hydrolase Cop9 signalosome complex subunit 5 (Csn5), augmented the band shift (Fig. 1c), suggesting that neddylation of Cul4a and Cul4b is activated during neuronal development. The level of native Cul4a increased until DIV10, but neddylated Cul4a did not change, resulting in a significant decrease in the ratio of neddylated vs. native Cul4a (Fig. 1d). Similarly, Cul4b neddylation also decreased at DIV10 (Fig. 1e). These results suggest that CRL4 is highly expressed and activated during the early stage of neurodevelopment rather than later stages, implying its developmental stage-specific functions.

Next, we performed immunocytochemistry of Cul4a and Cul4b. From DIV5 to DIV10, Cul4a immunoreactivity was evenly enriched in the nucleus and cytosolic compartments of neurons, colocalizing with Ddb1 immunoreactivity (Fig. 1f). In contrast, the immunoreactivity of Cul4b having a nuclear localization signal (NLS) in the N-terminus⁸, was highly enriched in the nucleus (Fig. 1g). Notably, at DIV5, a significant fraction of Cul4b was accumulated in the cytosol like Cul4a. According to the GenBank database, the rat Cul4b gene (gene ID: 361181) has three splicing-variants: Cul4b-1, -x1, and -x2. Cul4b-x2 does not have an N-terminal extension bearing an NLS sequence, unlike the other isoforms (Supplementary Fig. S2a). Expectedly, ectopically expressed Cul4b-x2 was mostly found in the cytosol rather than the nucleus, as expected, while other isoforms were strongly accumulated in the nucleus with a little cytosolic expression (Supplementary Fig. S2b). Cul4b-1 and -x2 transcript and protein levels were also found to be high on DIV5 but significantly lower on DIV10 (Supplementary Fig. S2c,d). Additionally, subcellular fractionation data showed that the level of cytosolic Cul4a and Cul4b is high at DIV5 with Ddb1 but decreased at DIV10 (Fig. 1h,i). We further performed immunohistochemistry for Cul4a and Cul4b in rat brains. Cul4a immunoreactivity showed cytosolic expression with Ddb1 in the cortical plate of the cerebral cortex of the embryonic brain, where developing neurons mainly exist (Fig. 1j). In cerebral cortex layer V of the adult brain, which contains the cell bodies of mature neurons, it was still enriched in both the nucleus and cytosol with Ddb1. In contrast, Cul4b exhibited cytosolic localization in the cortical plate of the embryonic brain, but obvious nuclear accumulation in the adult brain (Fig. 1k), consistently with the immunocytochemistry data. Taken together, we suggest that CRL4 is highly expressed and activated in the cytosol during early neuronal development, rather than at later stages (Fig. 1l).

Neuronal activity controls CRL4 via N-methyl D-aspartate receptor signaling

Neuronal activity tightly regulates neurodevelopmental processes including neuritogenesis, polarization via axon/dendrite specification, and final formation of the synapses^19,20. Thus, we hypothesized that neuronal activity might regulate the CRL4 expression or activity. We stimulated mature neurons (DIV10) by transient treatment with glutamate (Fig. 2a). While the Dbd1 protein level did not change by glutamate (Fig. 2b,c), Cul4a was significantly increased (Fig. 2b,d), resulting from an increase in the native form (Fig. 2b,e). Interestingly, co-treatment with AP5, a competitive antagonist of the N-methyl D-aspartate receptor (NMDAR), further increased the level of neddylated Cul4a but not the native form, reversing the glutamate-induced reduction of neddylation (Fig. 2e). However, neither NBQX, an AMPA/kainate receptor antagonist, nor LY341495 (LY), a metabotropic glutamate receptor antagonist, affected Cul4a expression (Fig. 2b,d,e). These results indicate that glutamate-evoked neuronal activity increases Cul4a protein level while limiting neddylation via NMDAR signaling. Unlike Cul4a, levels of total, neddylated, and native Cul4b gradually decreased until 5 hr after glutamate stimulation, and AP5 efficiently reversed this reduction in total and neddylated Cul4b, but neither NBQX nor LY (Fig. 2b,f,g). Additionally, glutamate induced nuclear accumulation of Cul4a along with Ddb1, and it was suppressed by AP5 (Fig. 2h). These results indicate that glutamate-evoked neuronal activity downregulates the expression, neddylation, and cytosolic localization of CRL4 via NMDAR signaling. We further examined whether direct NMDAR activation regulates CRL4. While NMDA did not affect Ddb1 (Fig. 2a,i,j), it increased the level of native, but not neddylated, Cul4a with a reduction in neddylation (Fig. 2i,k,l). Remarkably, memantine (MEM), an ion channel blocker of NMDAR, completely reversed the change in Cul4a expression (Fig. 2i,k,l). These findings indicate that, while NMDA increases Cul4a expression, it decreases neddylation and cytosolic localization. In contrast, Cul4b protein level was gradually reduced by NMDA until 5 hr, and it was efficiently reversed by MEM (Fig. 2i,m,n). Furthermore, NMDA also induced nuclear accumulation of Cul4a along with Ddb1, and it was reversed by MEM (Fig. 2o). Taken together, these results indicate that cytosolic CRL4 is controlled by cation influx via NMDAR signaling during neuronal activation. Considering that cytosolic expression and neddylation of Cul4a and Cul4b are higher in immature neurons than mature neurons, we postulated that CRL4 might function in the cytosolic compartment during the early neurodevelopmental stage.

CRL4 interacts with cytoskeleton-organizing proteins

To investigate the novel functions of CRL4 in neurodevelopment, we immunoprecipitated CRL4-interacting proteins by using Cul4a and Cul4b antibodies from the lysates of cultured neurons, and performed LC-MS/MS analysis of enriched proteins stained by Coomassie blue on SDS-PAGE (Fig. 3a,b). Expectedly, Cul4a-and Cul4b-interacting proteins highly overlapped, although some unique interacting proteins might also exist (Supplementary Table 1), implying high functional redundancy between Cul4a and Cul4b. Clustering analysis of gene ontology (GO) revealed that the pool size and GO terms of Cul4a/b-interacting proteins at DIV5 were smaller than those at DIV10, implying multi-functionalization of CRL4 during neurodevelopment. Particularly, we identified actin-filament protein (Actb), microtubule-associated proteins (Dcx and Dbn1), Sema3A signaling proteins (Crmp1 and Dpysl2/3/5), myosin proteins (Myh10 and Myo5a), and spectrin proteins (Sptan1 and Sptbn1) as co-interactors of Cul4a and Cul4b (Fig. 3c,d). These proteins are involved in neuronal morphogenesis including axon guidance and projection, by regulating the cytoskeletal organization (Fig. 3c,d,e). Previous studies demonstrated that in vitro cultured hippocampal or cerebral neurons undergo axon specification and extension with short dendrites to DIV4²¹. Thus, we hypothesized that CRL4 may work in the early neuronal morphogenesis.

CRL4 restricts axonal extension and branching in early neuronal development

To explore an impact of CRL4 in neuritogensis, we inhibited endogenous CRL4 by overexpressing the dominant-negative mutant of Cul4a (DN-Cul4a) or Cul4b (DN-Cul4b), which lacks Cullin, Rbx1/2-binding and neddylation domains²², with membrane-targeted GFP to visualize neurites at DIV2. After two days (DIV4), we found that Flag-tagged DN-Cul4a and DN-Cul4b were localized to the cytosolic compartment (Fig. S3a). Surprisingly, DN-Cul4a-expressing neurons had longer and more complex neurites than control neurons (Fig. 4a). Sholl analyses revealed that DN-Cul4a increased the complexity of distal axons and proximal dendrites, whereas DN-Cul4b showed such increase only in the dendrites (Fig. 4b). In addition, DN-Cul4a significantly increased the length of neurites, especially axons, but not dendrites (Fig. 4c,d). It also increased the number of neurites, particularly dendrites (Fig. 4e,f). However, DN-Cul4b, had an effect on only dendrite branching (Fig. 4a-f). Reversely, we overexpressed Cul4a and Cul4b with Ddb1 at DIV2. The overexpressed Cul4a was mainly localized in the cytosol, whereas Cul4b was mostly found in the nucleus with a little cytosolic expression (Supplementary Fig. S3b). Cul4a overexpression likely exhibited lower complexity in neurites, especially axons, than the Mock group (Fig. 5a,b). And it also significantly shortened the length of neurites containing axons and dendrites (Fig. 5c,d), and decreased axon branching (Fig. 5e,f). However, the nucleus-localizing Cul4b did not have a significant effect on neurite morphology (Fig. 5a-f). These results suggest that cytosolic CRL4 regulates the morphogenesis of neurites, especially axons, during the early neurodevelopment.

CRL4 antagonizes NMDA-induced neurite extension

NMDAR signaling is involved in the formation, outgrowth, and branching of axons and dendrites^23–26. Because NMDAR activation downregulates CRL4 in mature neurons (Fig. 2), we investigated whether NMDAR signaling also affects CRL4 in immature neurons, which exhibit a lower calcium response to NMDA than mature neurons²⁵. Notably, prolonged treatment of high-concentrated (100 µM) NMDA, which is non-toxic to immature neurons^25,27, did not affect the protein levels of Cul4a, Cul4b, and Ddb1 in DIV5 (Fig. 6a,b). However, neddylation of Cul4b was significantly increased by NMDA (Fig. 6a,c). Next, we investigated the role of CRL4 in NMDAR-mediated neuronal morphogenesis. Similar to a previous report²⁶, treatment with 1 µM NMDA dramatically elongated total neurite and axon neurite length, and significantly increased the complexity of the axon without additional neurite branching (Fig. 6d,e,g-j). However, overexpression of Cul4a or Cul4b effectively inhibited NMDA-induced extension and complexity in total neurites and axons (Fig. 6d,f-h). Considering that CRL4 activity is not correlated with NMDA in immature neurons, we suggest that CRL4 inhibits NMDA-induced axonal extension and complexity via an NMDAR-independent mechanism.

CRL4-Crbn complex controls the stability of Dcx protein

It has been well-known that Dcx facilitates axon and dendrite morphogenesis during neurodevelopment by stabilizing microtubules and regulating actin filaments^28–31. And Dcx protein is post-translationally regulated by the ubiquitination-proteasome system mediated by CRL3 or Mdm2 E3 ligase^12,32. Therefore, we postulated that CRL4 may also control stability of Dcx among the CRL4-interacting proteins. We reconfirmed that a small fraction of Dcx interacts with Cul4a and Cul4b by immunoblotting after immunoprecipitation (Fig. 7a), and further investigated where Dcx and CRL4 interact. Immunocytochemistry data showed that Dcx immunoreactivity is colocalized with Cul4a or Cul4b in proximal neurites around the soma (Fig. 7b). Furthermore, we performed a proximity ligation assay (PLA) to visualize the interaction between Dcx and CRL4. Interestingly, the amplified PLA signals of Cul4a-Dcx and Cul4b-Dcx showed punctate pattern on Tau-labeled proximal and distal parts of the axon, as well as around the soma (Fig. 7c). These results indicate that cytosolic Cul4a and Cul4b are associated with a subset of Dcx in axons. And next, to investigate how CRL4 affects Dcx protein, we overexpressed DN-Cul4a and/or DN-Cul4b in PC12 cells, which endogenously express the Dcx protein³³. The level of DN-Cul4b was significantly higher than that of DN-Cul4a, although bi-cistronically expressed GFP showed a similar level, indicating that DN-Cul4b is more stable. As expected, inhibition of cytosol-enriched Cul4a by DN-Cul4a significantly increased the levels of Dcx protein. However, DN-Cul4b had a similar effect to DN-Cul4a (Fig. 7d). This may be because a small fraction of DN-Cul4b localizes to the cytosol and it is more stable than DN-Cul4a (Supplementary Fig. S3a). Reasonably, the co-expression of DN-Cul4a and DN-Cul4b increased Dcx protein expression more dramatically. These results indicate that CRL4 negatively regulates Dcx protein stability.

Because DCAF is required to recruit the substrate to CRL4⁸, we screened DCAF for the association between CRL4 and Dcx. Through a literature survey and open-source tissue and intracellular expression data in the brain^34,35, we selected Cereblon (Crbn), which is associated with autosomal recessive intellectual disability³⁶, as a putative DCAF for Dcx. Crbn immunoreactivity was highly enriched in the cytosol around the soma and neurites of cultured neurons (Fig. 7e), as previously reported³⁷. In addition, the PLA signal of Dcx-Crbn showed a punctate pattern on phalloidin-labeled actin-filaments in neurites, similar to that of Dcx-Cul4a and Dcx-Cul4b (Fig. 7f). These data indicates that Crbn associates with Dcx in the neurites. To investigate whether Crbn-CRL4 complex affects Dcx protein stability, we overexpressed Crbn along with Cul4a + Ddb1 and Cul4b + Ddb1 in PC12 cells. Notably, Cul4a + Ddb1 or Cul4b + Ddb1 dramatically reduced the level of Dcx protein, and it was augmented by Crbn overexpression (Fig. 7g). Furthermore, we examined how Dcx regulation by Crbn-CRL4 complex impacts microtubule dynamics. The level of acetylated α-Tubulin, which is enriched in stable microtubules³⁸, was decreased by overexpression of Cul4a or Cul4b along with Ddb1, and it was potentiated by Crbn co-expression (Fig. 7h). On the other hand, DN-Cul4b or DN-Cul4a + DN-Cul4b increased the level of acetylated α-Tubulin (Fig. 7i). These results indicate that CRL4 regulates microtubule stabilization. In conclusion, we suggest that CRL4 regulates axonal morphogenesis in the early neurodevelopmental stage by restricting Dcx-mediated microtubule stabilization (Fig. 7h).

In this study, we demonstrated that cytosolic CRL4 plays a neuromorphogenic role in neurodevelopment. Cul4a, Cul4b, and Ddb1, the three main components of CRL4, are highly expressed in the cytosolic compartment of developing neurons. Previous studies demonstrated that the Cul4a protein localizes to the cytosolic compartment, while Cul4b is prone to accumulate in the nucleus despite their high identity in protein sequence and functional redundancy⁸. This difference may be attributed to the NLS-bearing N-terminal extension of Cul4b. Remarkably, our data showed stage-specific cytosolic expression of Cul4b in developing neurons. Although the Cul4b-1 and -x1 are major products of the Cul4b gene in the adult brain, as previously reported³⁹, Cul4b-x2 without NLS-bearing N-terminus seems to be responsible for the cytosolic function of Cul4b. When ectopically expressed, it mainly localizes to the cytosol unlike other isoforms, and its mRNA and protein levels exhibit transient expression in developing neurons. Cell-type- and time-specific alternative splicing have been known to be involved in neuronal differentiation, neuritic outgrowth, and synaptic formation during brain development⁴⁰. For example, the pre-mRNA of postsynaptic density protein 95 undergoes splicing-dependent decay by timely expression of polypyrimidine tract-binding proteins, Ptbp1 and Ptbp2, in the early neurodevelopmental stage⁴¹. Thus, we believe that Cul4b pre-mRNA may be post-transcriptionally regulated by stage-specific alternative splicing during neurodevelopment. In addition, we showed that the neddylation of Cul4a and Cul4b was transiently enhanced in developing neurons. A previous study reported that Cul4b-x2 is more neddylated in the adult mouse brain than Cul4b-1 and Cul4b-x1, despite having the lowest expression³⁹. These results suggest that cytosolic CRL4 is highly functional during the early neurodevelopmental stage.

Neuronal activity is essential for neuronal development, survival, maturation, synaptic plasticity, and regeneration^19,20. Our results show that NMDA-evoked neuronal activation downregulates CRL4 activity in mature neurons but not in immature neurons. In particular, NMDAR-mediated cation influx dramatically reduced the neddylation of Cul4a and Cul4b. Consistently, Cul4a/Cul4b neddylation levels were lower in mature neurons than in immature neurons. This process may result from the inhibition of neddylating protein including Nae1, or the activation of deneddylating molecules such as Cop9 signalosome and Cullin-associated NEDD8-dissociated protein 1⁸. Although no direct evidence of a molecular link between NMDA signaling and CRL neddylation has been proposed, previous studies may provide some insight into this issue. Intracellular calcium influx can activate the NF-κB signaling pathway in response to NMDA-evoked excitotoxicity^42,43. A recent report demonstrated that Csn5 is activated via phosphorylation by the IKK complex, an NF-κB signaling molecule⁴⁴. These findings raise the possibility that NMDA-mediated calcium signaling inhibits CRL neddylation, although further research is needed. In addition, our data showed that glutamate/NMDA stimulation reduced the cytosolic localization of Cul4a by inducing nuclear accumulation in mature neurons. Thus, we suggest that the cytosolic function of Cul4a is down-regulated under neuronal stimulation. Reversely, nuclear accumulation of Cul4a may be involved in the protective response against DNA damage. Upon UV exposure, the Cul4a-Ddb1 and/or Cul4b-Ddb1 complex is recruited to damaged DNA foci of the nucleus by Ddb2, which recognizes several substrates including histone H2A for nucleotide excision repair and other DNA damage responses as a DCAF⁴⁵. Similarly, Cul4a is translocated into the nucleus and inhibits Parp1-mediated cell death under oxidative stress⁴⁶. In neurons, glutamate/NMDA stimulation can also induce DNA damages such as oxidation and single/double-strand breakage, resulting in activity-induced gene expression or neurodegeneration^47,48. Moreover, the Cul4a-Ddb1-Ddb2 complex is involved in base excision repair of oxidized DNA⁴⁹. These reports imply that glutamate/NMDA-induced nuclear accumulation of the Cul4a-Ddb1 complex may play a role in protective repair mechanisms against DNA damage. Thus, we believe that CRL4 may function differently in the cytosol and nucleus under neuronal stimulation.

We first identified CRL4-interacting proteins in neurons. Despite the use of different experimental models, 29 of the 67 potential interactors have already been proposed as Cul4a-or Cul4b-interacting proteins based on open-source interactome data⁵⁰ (Supplementary Table 1). This result implies that our experiment is reliable and that the interaction may be independent of cell-type. Cytoskeletal constituents such as actin, tubulin, and spectrin; cytoskeleton-regulating proteins including Dcx, Dbn1, and Tmod2; and motor proteins such as Myo5a, Myh10, and Spire1 are highly associated with CRL4, coincidently with the cytosolic expression of Cul4a and Cul4b. Furthermore, in DIV10, RNA/DNA-binding molecules such as Tdp-43, Hnrnpd, Hnrnpf, Hnrnpg, Hnrnpk, Hnrnpl, Macroh2a2, Edf1a1, Ilf2, and Ell, as well as mitochondrial proteins such as Pdha1, Sucla2, Acat1, Atp5f1a, Uqcrc2, Slc25a3-4, Ywhaz, and Pck2, were identified as Cul4a-and Cul4b-interactors as well as cytoskeleton-regulating proteins. Considering that Cul4b is involved in dendritic spine formation in hippocampal neurons¹⁵, our data suggest that CRL4 may play a more versatile role in mature neurons.

Our results also revealed that cytosolic CRL4 functions as a negative regulator of axonal morphogenesis. Genetic perturbation of Cul4a, which is prone to cytosolic localization, has a stronger effect on axonal extension and branching than nucleus-enriched Cul4b. However, upon NMDA treatment, Cul4b exhibited a similar inhibitory effect against NMDA-induced axonal extension as Cul4a. It may result from the upregulation of its neddylation by NMDA. Considering that NMDA does not affect the expression and neddylation of Cul4a and Cul4b in the immature neurons (DIV5), CRL4 seems to work antagonistically and/or independently to NMDA in axonal morphogenesis. According to our data, CRL4 dysfunction could induce deregulated hyper-extension or over-sprouting of axons. However, axonal morphological defect has never been reported in Cul4a knockout, Cul4b knockout, and even Ddb1 knockout mice^15,51−55, although the previous studies have not focused on this issue. And even, Cul4b has been identified as a positive regulator of neurite outgrowth and branching^15,56. Cul4b mediates NGF-induced neurite outgrowth in PC12 cells by targeting Wdr5, which epigenetically silences neuronal genes as a core subunit of the histone H3 lysine 4 methyltransferase complex⁵⁶. Cul4b-knockout male mice consistently exhibit a significant X-linked intellectual disability phenotype, owing to reduced complexity and spine density of dendrites in hippocampal neurons¹⁵. However, Cul4a-knockout mice show no intellectual disability phenotype or neurological defects⁵¹, implying that Cul4a-irreplaceable function of nucleus-localizing Cul4b in the nervous system. Thus, we suggest that the inconsistency between our study and previous studies may result from the differential function of nuclear Cul4b-scaffolded CRL4 vs. stage-specific cytosolic Cul4a/Cul4b-scaffolded CRL4 in neuritogenesis.

DCX genetic mutations, like CUL4B, have been linked to X-linked intellectual disability and epilepsy⁵⁷. As CRL4 regulates the stability of Dcx protein, CRL4 and Dcx may share the same neuritogenesis signaling pathway. Dcx is also controlled by other E3 ligases. Mdm2 restricts the level of Dcx via the ubiquitination-proteasome degradation system, thereby inhibiting dendritic spine morphogenesis in olfactory bulb interneurons³². Klhl15-CRL3 complex targets Dcx, resulting in restriction of neuritogenesis¹². This implies that CRL3 and CRL4 may share substrates and have overlapping functions in neuritogenesis. Coincidently, Keap1-guided CRL3 and Wdr23-guided CRL4 independently targeted Nrf2, a transcription factor that mediates the stress response⁵⁸. In addition, Crbn-bound CRL4 and Fbxo7-bound CRL1 inhibit voltage- and calcium-activated big-conductance potassium channels via ubiquitination⁵⁹. These reports suggest that CRLs may reciprocally and complementary function to each other. Therefore, inhibition of CRL4 may not be sufficient to induce neurodevelopmental defects, because Dcx can be ubiquitinated by other CRLs or E3 ligases.

Lastly, the DCAFs involved in neurodevelopment, neurophysiology, and neuropathology are largely unknown. Considering that CRL4 has been proposed as a platform for proteolysis-targeting chimera protein degraders that remove aberrant or toxic proteins via chemical-linked ubiquitination⁶⁰. Further studies on neuronal DCAF may provide novel strategies against neurological disorders and neurodegenerative diseases.

Conflict of Interest

There are no conflicts of interest to declare

Author contributions

T.S, B.C. and C.M. mainly contributed to the study conception and design. T.S. and B.C. performed the experiments, analyzed the experimental data, and wrote first draft of the manuscript under supervision of C.M.. J.K. and Y.L. provide experimental materials and commented on manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Mr. Kwangsoo Kim for statistical advice and analysis. This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2020M3A9D8038658 and 2020R1A2C2005174) and by the Ministry of Education (2020R1A6A1A03040516).

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Cullin-RING E3 ubiquitin ligase 4 controls axonal morphogenesis during neuronal development

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Abstract

Figures

Introduction

Materials And Methods

Experimental animals

Rat primary cerebral neuron culture and PC12 cell culture

Chemicals

Immunoblotting and Antibodies

Immunocytochemistry and proximity ligation assay

Immunohistochemistry

Fractionation of nucleus and cytosol

Immunoprecipitation

LC-MS/MS

DNA Constructs and gene transfection

Quantification of mRNA level

Quantification of neurite morphology

Statistical analysis

Results

Cul4a and Cul4b are up-regulated during early neuronal development

Neuronal activity controls CRL4 via N-methyl D-aspartate receptor signaling

CRL4 interacts with cytoskeleton-organizing proteins

CRL4 restricts axonal extension and branching in early neuronal development

CRL4 antagonizes NMDA-induced neurite extension

CRL4-Crbn complex controls the stability of Dcx protein

Discussion

Declarations

Author contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

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