Investigating the Role of NCAM-1 and Profilin-2 in Synaptic Re-modeling During Neural Development in C. elegans

ABSTRACT

Synaptic remodeling is the process of rewiring neural connections during development. This process is essential for the creation of functioning neural circuits in the brain; disruptions in this mechanism have been tied to several neurological disorders like attention deficit hyperactivity disorder, autism spectrum disorder, and epilepsy. To investigate the molecular pathways that regulate synaptic remodeling, this study investigated the roles of two proteins in C. elegans: NCAM-1, a cell adhesion protein with key roles in synaptogenesis and maintenance, and Profilin-2 (PFN-2), an actin-binding protein which helps shape the neurons through actin dynamics. Previous work has shown that NCAM-1 mediates synaptic remodeling in developing GABAergic neurons, and additional findings suggest that NCAM-1 may interact with PFN-2 intracellularly. To test the role of these proteins in synaptic remodeling, we used the DD GABAergic motor neurons in C. elegans because they undergo a temporally and transcriptionally regulated remodeling period during early larval development. Using live-cell confocal imaging to track remodeling of synaptic proteins, we confirm that ncam-1 null mutants showed clear synaptic remodeling defect. On the other hand, the pfn-2 null mutants did not display any significant synaptic remodeling abnormalities when compared to the wildtype animals, which suggests that other profilins may compensate for the loss of PFN-2.

INTRODUCTION.

Neurological Disorders.

Neurological and neuropsychiatric disorders such as autism spectrum disorder (ASD), schizophrenia, epilepsy and neurodegeneration are increasingly understood as disorders of circuit wiring and plasticity rather than purely a consequence of “cell loss.” Researchers have been able to observe changes in the structure of the synapse as well as the number of synapses across neurological disorders. These modifications include variability in neuron morphology, disturbance in the excitatory and inhibitory signals, and irregularities in how a synapses are weakened or strengthened with activity [1].

Risk genes for neurological disorders often encode proteins involved in synapse assembly, function, or maintenance [2]. Many of these genes regulate key components of synaptic structure, such as cell-adhesion molecules, scaffolding proteins, and presynaptic machinery. Others influence the cytoskeletal dynamics that stabilize and remodel synapses over time.

Synaptic Remodeling.

Synaptic remodeling is the rearrangement of synaptic connections after they have initially formed. Examples of remodeling includes the formation and stabilization of new synapses, weakening or removal of other synapses, and relocating pre-existing synapses onto new targets during development [3]. Remodeling is coordinated by guidance and adhesion molecules and cytoskeletal dynamics. Activity-dependent mechanisms may determine which synapses are retained versus removed to tune circuit function and preserve excitatory/inhibitory balance [4]. An excitatory signal is what increases the chance of a neuron’s action potential; too much neuronal excitation can cause hyperactivity, epilepsy, and seizures – all symptoms of autism spectrum disorder. Inhibitory signals make the neurons less likely to release neurotransmitters; too much inhibition can cause a neuron to become underactive, which can lead to memory loss and delayed/impaired learning. Cell-adhesion molecules are thought to act as “coordinates” that guide synapse matching and relocation. Profilins regulate actin dynamics and endocytosis [5]. Actin filament assembly and disassembly generate the mechanical force needed for membrane movement. When these pathways are disturbed, synapses can be misplaced or unstable. Remodeling unfolds on defined developmental timelines and can be imaged in vivo.

Molecular View of the Synapse.

Synapses are the specialized connection points where neurons communicate with their post-synaptic targets, allowing information to flow through the nervous system. These connections operate through either electrical signaling via gap junctions that directly pass ions between cells, or chemical signaling via neurotransmitters released from one neuron and detected by another. Chemical synapses are made up of two major components: the presynaptic terminal, which releases neurotransmitters, and the postsynaptic site, which receives and responds to them [6].

Structurally, synapses are organized by a complex network of scaffolding proteins, cell-adhesion molecules, and cytoskeletal elements that stabilize the connection and ensure precise signaling. This architecture not only maintains synaptic integrity but also supports synaptic plasticity, the process through which synapses strengthen, weaken, or remodel over time. Synaptic plasticity is essential for learning, memory formation, and refinement of neural circuits during development [6].

Studying Synaptic Remodeling in C. elegans.

The nematode, Caenorhabditis elegans (C. elegans), provides a useful system to study synaptic remodeling. C. elegans is transparent and genetically tractable, which makes it readily accessible for genetic analysis and live-cell imaging [7]. Other benefits include a fully sequenced genome, complete neuronal wiring diagram and gene expression atlas of the nervous system [8], as well as established genetic tools such as RNA interference (RNAi) libraries, transgenic reporter lines, and mutant libraries to enhance functional genomics studies [8].

To investigate the mechanisms regulating synaptic remodeling, CRISPR gene editing was used to fluorescently label a presynaptic protein, RAB-3, with GFP, enabling live-cell imaging of RAB-3 at synapses [3]. This technique enables researchers to examine how mutations affect synapse organization and neuronal connectivity.

Neural Cell Adhesion Molecule (NCAM-1) and Profilin-2 (PFN-2).

NCAM-1 is a highly conserved cell-adhesion protein that promotes synapse formation, regulates synaptic strength, and contributes to memory formation [9 -10]. Disruption of NCAM-1 has been linked to different neurological disorders such as Alzheimer’s, schizophrenia, and bipolar disorder [11]. Additional work has shown that NCAM-1 has key roles in memory and learning. One study investigated the role of NCAM1 in both human and C. elegans (Figure 1). They showed that in C. elegans NCAM-1 levels increased after conditioning and that worms lacking ncam-1 could form short-term but not long-term memories; rescuing the gene restored memory [12].

Profilins are conserved proteins which regulate cytoskeletal organization through signaling and by promoting actin polymerization and turnover [13]. Different profilins are expressed across systems, but profilin-2 (PFN-2) is primarily expressed in the brain, where it regulates actin dynamics and endocytosis [5]. PFN-2 catalyzes the exchange of ADP for ATP on actin monomers to promote actin polymerization.

Researchers have found that the loss of NCAM1 disrupts actin organization within synapses, leading to impaired cytoskeletal dynamics and synaptic instability [12]. Additionally, a 2020 study showed that NCAM-1 and PFN-2 physically interact at a conserved amino acid residue on the NCAM-1 intracellular tail [14]. Due to this, PFN-2 may function downstream of NCAM1 through this interaction to mediate synaptic remodeling.

Remodeling of Dorsal (DD) Motor Neurons.

During development, C. elegans GABAergic neurons undergo synaptic remodeling in which presynaptic sites shift from ventral to dorsal regions; DD ventral synapses are eliminated, and new synapses are formed on the dorsal side (Figure 2) [3]. Disruption in the DD remodeling process can cause an imbalance of inhibitory GABAergic signaling to muscles which can result in worms curling up and impairing movement.

Study Applications.

The focus of this study is investigating the mechanism by which NCAM-1 regulates synaptic remodeling. Although previous work has shown that NCAM-1 mediates re-modeling in C. elegans, the downstream mechanism is not well understood. Because actin dynamics is important for synaptic remodeling, and a previous paper showed that mammalian PFN2 interacts with a conserved NCAM1 intracellular domain, we hypothesize that PFN-2 is required for NCAM-1-mediated synaptic remodeling in C. elegans DD neurons. Therefore, the objective of this study is to determine whether PFN-2 is required for synaptic remodeling in C. elegans DD neurons, based on the hypothesis that the absence of PFN-2 will disrupt synaptic remodeling be-cause NCAM-1 requires PFN-2 to mediate actin-dependent structural changes.

MATERIALS AND METHODS.

C. elegans Strains and Maintenance.

Wildtype (WT) N2 worms and mutant strains (ex. null alleles pfn-2(ok458), ncam-1(syb6584)) were maintained on nematode growth media (NGM) agarose plates seeded with E. coli OP-50 and maintained at 23°C [15]. pfn-2(ok458) allele purchased from the Caenorhabditis Genetics Center (CGC).

Genetic Crossing.

To test the effect of ncam-1 and pfn-2 loss of function on synaptic remodeling, we used genetic lines containing endogenously GFP-tagged RAB-3, which fluorescently labels a presynaptic protein for visualization. Wild-type and ncam-1 null mutants were used as controls. For this study, we crossed a pfn-2(ok458) null mutation into the GFP:RAB-3 line. New strains were built using standard genetic crosses [15] and confirmed through genotyping.

Genotyping.

Lysis buffer was made by adding 1 μL of 35 mg/mL ProteinaseK per 100 μL of lysis buffer solution (50 mM KCl10 mM Tris (pH 8.3)2.5 mM MgCl20.45% Nonidet P-400.045% Tween-20). Worms from each plate were carefully picked and transferred directly into the lysis buffer in a PCR tube. The tubes were then briefly spun to ensure the worm settled at the bottom. Samples were immediately incubated in a thermocycler at 60 °C for one hour, then 95°C for fifteen minutes to yield lysate DNA

GoTaq (PCR).

PCR was conducted using the GoTaq DNA Polymerase Kit (Promega). 2.5 μL of DNA lysate was added to separate PCR tubes and the Taq-PCR reaction mixture (base on kit protocol) was combined with gene specific primers to amplify either ncam-1 or pfn-2 mutations for genotyping analysis. The PCR containing reaction mix and DNA lysate were incubated in a thermocycler based on protocol-define settings.

Gel Electrophoresis.

The DNA product was separated on a 1% agarose gel containing 3 uL of ethidium bromide in a 1× TAE buffer. Gels were run at 120V for 20 minutes. The gel was then examined using a Bio-Rad Gel Doc XR+ imaging system. Band sizes were compared to a 1kb+ DNA ladder (New England Biolabs) to confirm successful mutations. Each primer set labels separately sized wildtype and mutant DNA fragments for identifying heterozygous from homozygous animals. Successful homozygotes were isolated and used for further experiments.

Mesh Synchronization (Hour Post-Hatch [hph] Image prep).

Strains were grown on 3 150mm agarose plates under standard conditions. Worms were washed off the 150 mm plates using 5 mL of MilliQ (pure water) per plate and collected into a 50 mL conical tube. This suspension was then centrifuged at 2.5k rpm for 2.5 minutes. Animals were pelleted to the bottom, and the supernatant was aspirated out of the tube; the samples were washed three times under these setting to remove any bacteria from the collected animals. Bleach solution (7.5ml H2O, 2.5ml 10% bleach, 0.5ml 10N NaOH) was added to kill off all the worms except the embryos. The mixture was nutated for approximately 4 minutes and monitored by microscopy to ensure partial lysis of adults without damaging embryos. The reaction was stopped with 30 mL of M9 buffer, and the sample was centrifuged and washed three times with M9.

The embryos were then resuspended in 5 mL of M9 and pipetted onto an 18 μm mesh that was mounted in an 8-inch embroidery hoop and placed onto liquid food, allowing embryos to hatch and crawl through the mesh for 1 hour. The mesh was then rinsed with ~15 mL of M9 to recover synchronized L1 larvae. The larvae were moved to a fresh plate and allowed to grow. Finally, they were prepped for imaging at the 17 hph mark to visualize synaptic remodeling 17hph was selected as a mid-late remodeling timepoint for comparison across genotypes.

Preparing the worms for imaging.

~30 worms were transferred onto a microscope slide containing an anesthetic mixture to immobilize them. The mixture consisted of 5 µL of 100 mM muscimol and 5 µL of a time-lapse anesthetic solution (1% tricaine and 0.5 M levamisole). A coverslip was then sealed in place with wax, and the samples were prepared for imaging.

Live-cell Imaging and Analysis.

Live-cell imaging was performed using a Nikon A1R confocal microscope. NIS Elements software was used for imaging. For each animal, Z-stack images were acquired on 60x oil immersion objective using laser excitation settings for the GFP and mCherry. Nikon Analysis Software (NIS-Elements) was used for image processing and analysis. Z-stack images were converted to maximum-intensity projections (Max-IP). The second and third DD neurons were used for analysis. To analyze signal localization, line profiles were drawn over the ventral and dorsal nerve cord for each neuron. During the remodeling period, synapses are removed from the ventral nerve cord and new synapses are formed on the dorsal nerve cord. Line profile data was used to calculate average fluorescence intensity of RAB-3 signal. This was normalized against background fluorescence intensity and remodeling was quantified as percent of signal localized ventral/dorsal. GraphPad PRISM analysis software was used to conduct ANOVA testing to compare multiple groups (WT, ncam-1, and pfn-2). Fiji/ImageJ software was used for creating representative images.

RESULTS.

Ventral and dorsal synaptic signals were compared between wild-type and mutant animals to assess differences in synaptic remodeling (Figure 3). A one-way ANOVA showed a significant difference between WT and ncam-1, validating the control. However, no significant difference was observed between WT and pfn-2, indicating that pfn-2 animals remodel comparably to WT.

DISCUSSION.

Wild-type (WT) animals were used as a positive control to provide a baseline comparison of successful synaptic remodeling. ncam-1 mutant animals were used as a negative control, showcasing defective remolding that is correlated with the loss of NCAM-1 . These control groups confirmed that the assay could detect defective remodeling when a required molecule (e.g., ncam-1) is absent, thus validating results observed from the pfn-2 mutant animals

A one-way ANOVA confirmed that the extent of remodeling in pfn-2 mutants was comparable to that of WT animals (Figure 3). In contrast, both WT and pfn-2 animals showed significantly different remodeling compared to ncam-1mutants. This outcome suggests that, unlike NCAM-1, PFN-2 is not essential for the remodeling process under the conditions tested. One possible explanation for the absence of a remodeling defect in the pfn-2 mutant is that other members of the profilin family may compensate for the loss of PFN-2. PFN-1, for example, is expressed throughout the body of C. elegans, whereas PFN-2 is enriched in the nervous system, and both function as actin-binding proteins that regulate actin filament dynamics.

CONCLUSION.

This focus of this study was to examine the role that the actin binding protein PFN-2/Profilin in a synaptic remodeling event in the C. elegans nervous system. Using genetic crossing, mutant C. elegans lacking the PFN-2 protein were combined with a strain in which the presynaptic protein RAB-3 was labeled with fluorescent GFP-tag for live-cell imaging to determine if the absence of PFN-2 disturbed the synaptic remodeling process. Live imaging of the GFP::RAB-3 showed that pfn-2 mutants remodeled like wild-type animals, suggesting PFN-2 is not essential for NCAM-1–mediated remodeling. These findings refine understanding of how actin-regulating proteins contribute to neural plasticity, indicating that other profilin family members or parallel pathways may compensate for PFN-2 loss.

Future work will test if the physical interaction between NCAM-1 and PFN-2 is necessary for proper synapse disassembly and formation, as disrupting this interaction may impair these processes. In addition, future studies should include testing different profilins like PFN-1 because it is expressed all over the body and may contribute to synaptic remodeling in ways that differ from PFN-2. Future studies should also investigate whether PFN-1 or other profilins compensate for PFN-2 loss using double-mutant analyses (pfn-1; pfn-2) and examine CRISPR-edited NCAM-1/PFN-2 binding mutants to test the necessity of their physical interaction. These steps are crucial for clarifying the molecular pathways that underlie synaptic remodeling and NCAM-mediated neural development.

ACKNOWLEDGEMENTS.

The author would like to thank the Miller Lab at Vanderbilt, specifically Dr. David Miller III and Ph.D. student Casey Gailey for mentoring her during the summer and making this project possible. She would also like to thank the REHSS program for allowing her to participate in this amazing experience. Supported by NIH grants to CG (F31MS134292) and DMM (R01NS106951).

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Posted by buchanle on Thursday, May 14, 2026 in May 2026.

Tags: C. elegans, NCAM-1, neural development, Profilin-2, synaptic remodeling