ADCY9 Regulates Neural Stem Cells Via Mitofusin-1 to Maintain Planarian (Dugesia japonica) Cephalic Ganglia Regeneration
Xinrui Wang, Sitong Hu, Ruijia Zhang, Xinlu Han, Lili Gao, Fengtang Yang, Zhonghong Cao, Hui Zhen
TL;DR
ADCY9 helps planarians regenerate their brains by controlling neural stem cells and working with Mitofusin-1.
Contribution
ADCY9's role in planarian neural regeneration and its interaction with Mitofusin-1 is newly identified.
Findings
ADCY9 knockdown disrupts brain regeneration in planarians, causing nerve cord loss and reduced neuron differentiation.
Mitofusin-1 RNAi rescues regeneration defects caused by ADCY9 knockdown, showing their functional link.
ADCY9 downregulation leads to 499 differentially expressed genes linked to neurodegenerative diseases.
Abstract
What are the main findings? ADCY9 upregulation is essential for planarian cephalic ganglia regeneration after amputation, and ADCY9 knockdown impairs this process.ADCY9 RNAi inhibits neural stem cells and neuronal differentiation during cephalic ganglia regeneration. ADCY9 upregulation is essential for planarian cephalic ganglia regeneration after amputation, and ADCY9 knockdown impairs this process. ADCY9 RNAi inhibits neural stem cells and neuronal differentiation during cephalic ganglia regeneration. What are the implications of the main findings? Mitofusin-1 RNAi rescues cephalic ganglia regeneration defects caused by ADCY9 knockdown.These findings reveal a critical role of the ADCY9–mitofusin-1 axis in planarian neural regeneration. Mitofusin-1 RNAi rescues cephalic ganglia regeneration defects caused by ADCY9 knockdown. These findings reveal a critical role of the…
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Figure 7- —National Natural Science Foundation of China
- —Space Application System of the China Manned Space Program
- —Natural Science Foundation of Shandong Province
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Taxonomy
TopicsPlanarian Biology and Electrostimulation · Developmental Biology and Gene Regulation · Plant and Biological Electrophysiology Studies
1. Introduction
The nervous system is crucial for governing physiological activities and mediating sensory–cognitive functions, yet its post-injury regenerative capacity is extremely limited, leading to motor, cognitive, and language deficits [1]. Neurodegenerative diseases like Alzheimer’s (AD), Parkinson’s (PD), and amyotrophic lateral sclerosis (ALS) are increasingly prevalent due to global aging, severely impairing patients’ quality of life and imposing heavy family burdens [2,3]. However, their underlying mechanisms remain unclear, with clinical interventions only relieving symptoms. Thus, in-depth studies on neural regeneration are urgently needed, and planarians—endowed with remarkable regenerative abilities, simple maintenance, and high genetic homology with humans—have emerged as an ideal model for exploring in vivo neural regeneration mechanisms. Planarians can regenerate multiple tissues (epidermis, muscles, nervous system, etc.) after amputation, and recent advances (e.g., single-cell sequencing and RNAi) have uncovered key mechanisms in their neural regeneration: notum^+^ and frizzled 5/8-4^+^ muscle cells guide newborn neuron distribution and circuit assembly, adult pluripotent stem cells (neoblasts) rapidly repopulate after injury, and the ERK pathway mediates “ERK waves” to activate regeneration programs [4,5,6,7,8,9,10,11]. Furthermore, genomic sequencing has revealed a high degree of homology between planarian and human genes [12,13]. Notably, planarian neural regeneration involves both morphological and functional recovery, highlighting its value in revealing conserved neural regeneration mechanisms.
The adenylyl cyclase (ADCY) family is made up of key metabolic enzymes responsible for intracellular generation of the second messenger molecule cyclic adenosine monophosphate (cAMP), which acts as a central signaling hub in the central nervous system. ADCY9 (adenylyl cyclase type 9) is involved in a variety of cell signaling pathways by regulating the level of cAMP and plays a complex and central role in cancer development, nervous system function and metabolic regulation [14,15].
ADCY9 is a key factor in the regulation of neuropathic pain. In rat models of sciatic nerve injury, miR-142-3p expression is significantly upregulated; this microRNA directly targets and represses ADCY9, consequently reducing the production of its downstream effector cyclic adenosine monophosphate (cAMP). This suppression impairs the activity of the cAMP/AMPK signaling pathway [16]. The ensuing molecular cascade elevates the levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which exacerbate neuropathic pain pathogenesis. Notably, inhibition of miR-142-3p to restore ADCY9 functionality or direct activation of the cAMP signaling axis can effectively reverse this pathological process and alleviate pain phenotypes [17]. ADCY9 plays a neuroprotective role in alleviating neuropathic pain, but its function in planarian neural regeneration is unreported.
Mitofusin-1 (Mfn1), an outer mitochondrial membrane protein, modulates mitochondrial dynamics to govern cell metabolism, immune responses and stress responses, with its expression and post-translational modifications directly impacting mitochondrial function and contributing to the pathogenesis of multiple diseases. In alveolar macrophages, Mfn1 deficiency blocks mitochondrial stress-induced hyperfusion, reduces reactive oxygen species production and lipid peroxidation, enhances antioxidant activity, and thus alleviates Streptococcus pneumoniae-induced lung inflammation and tissue damage [18]. In acute myeloid leukemia, IL6 upregulates Mfn1-mediated mitochondrial fusion to promote oxidative phosphorylation and chemoresistance, while Mfn1 knockdown or anti-IL6 antibodies reverse these effects to boost chemotherapy efficacy and prolong survival [19]. In intervertebral disc degeneration, Mfn1 collaborates with Mfn2 in responding to inflammatory signals (with Mfn1 showing a more prominent inflammatory response that induces mitochondrial fragmentation and extracellular matrix degradation) [20], and vitamin E treatment can suppress Mfn1 expression to preserve mitochondrial structure/function and extracellular matrix integrity. In osteoarthritis, PARP12- and ISG15-mediated ISGylation of Mfn1 inhibits its ubiquitination, and SUMOylation impairs PINK1/Parkin-dependent mitophagy and accelerates cartilage degradation, a process amplified by IRF1-upregulated PARP12 [21]. However, the role of Mfn1 in neural regeneration, especially in central nervous system reconstruction of highly regenerative models such as planarians, remains an uncharted field warranting further research.
In this study, we examined the spatiotemporal expression pattern and regulatory function of ADCY9 during planarian brain regeneration. The results demonstrated that the downregulation of ADCY9 resulted in abnormal brain regeneration in planarians, characterized by partial loss of the nerve cord, reduced numbers of collateral branches, significant inhibition of the regeneration and differentiation of multiple neuron types. To further elucidate the underlying mechanism, RNA-seq revealed that the downregulation of ADCY9 led to 499 differentially expressed genes, with KEGG enrichment pathway analysis indicating significant associations with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Double interference RNAi experiments demonstrated that simultaneous knockdown with ADCY9 and Mitofusin-1 significantly restored neural regeneration. Collectively, our results indicate that ADCY9 might promote the comprehensive reconstruction of neural structure by hierarchically regulating the regeneration intensity through negative regulation of the downstream inhibitory factor Mitofusin-1.
2. Materials and Methods
2.1. Materials
Planarian specimens (Dugesia japonica) were collected from Yiyuan County, Zibo, Shandong Province. Laboratory cultivation was conducted in Lushan spring water at 20 °C, with consistently clean water and a stable temperature maintained. Fresh beef liver served as the primary feed. Prior to experiments, planarians were fasted for one week. Each planarian’s head was amputated posterior to the eyespots and transferred to fresh water for regeneration. Samples were collected at five regeneration stages: 1, 3, 5, 7, and 10 days post-amputation (dpa).
2.2. Acquisition of ADCY9 Gene
Samples were ground in liquid nitrogen and homogenized with TRIzol reagent. PCR amplification was performed with cDNA of planarian as a template and corresponding primers. The reaction program was as follows: 94 °C pre-denaturation for 5 min; 35 cycles of 94 °C denaturation for 50 s, 55 °C gradient temperature denaturation for 60 s, 72 °C extensions for 60 s; and 72 °C extensions for 10 min. The PCR products were analyzed by gel electrophoresis and the optimal amplification temperature was selected according to the results.
2.3. RNA Interference (RNAi)
(1)Generation of the PCR template
PCR system: ddH_2_O,10 × PCR buffer, dNTP mix, T7 primers, and Taq DNA polymerase; reaction program: 95 °C pre-denaturation for 5 min; 35 cycles of 94 °C denaturation for 30 s, 55.0 °C annealing for 60 s, and 72 °C extension for 90 s; 72 °C extension for 10 min.
(2)Synthesis of dsRNA
The PCR template, DEPC water, 10 × T7 Reaction buffer, ATP, CTP, GTP, UTP, and T7 Enzyme Mix were mixed and incubated at 37 °C for 2–4 h, then at 75 °C for 5 min, followed by cooling at room temperature.
(3)DNA and ssRNA elimination and purification
Components: dsRNA, DEPC water, 10× Digestion Buffer, DNAse, and RNase. Mix and centrifuge at 37 °C for 1 h. Components: dsRNA, 10× Binding Buffer, DEPC water, and 100% ethanol. Reverse the mixture and centrifuge at 12,000 rpm for 2 min. Remove the supernatant, add Wash Solution, and centrifuge at 12,000 rpm for 2 min, repeating this process multiple times. Remove the supernatant and perform dry centrifugation at 12,000 rpm.
(4)Planarian interference treatment
The purified dsRNA was diluted to a concentration of 40 ng/μL. Planarians were incubated in the dsRNA solution at 19 °C for 6 h overnight. The procedure was repeated 5 times, and the treated planarians were then used for head amputation regeneration assays or in situ hybridization. Green fluorescent protein (GFP) dsRNA was administered to control planarians.
2.4. Quantitative Detection of ADCY9 Expression: qPCR
The cDNA samples were divided into five groups based on the number of regeneration days, with GAPDH as the control. qPCR was performed using the corresponding primers (Table S2) with SYBR Green mix, cDNA, forward primer, and reverse primer in a ratio of 5:4:0.5:0.5. The reaction protocol was: 95 °C denaturation (10 s), 55 °C annealing (10 s), and 72 °C extension (10 s), with a total of 40 cycles.
2.5. Whole-Mount In Situ Hybridization (WISH)
Samples were treated with 5% N-acetylcysteine (NAC) for 10 min, then fixed in 4% paraformaldehyde (PFA) at room temperature for 30 min. After PBST washing, they were treated with Reduction Buffer in a 37 °C water bath for 10 min. Samples were dehydrated through a methanol gradient (50% and 100%) and bleached overnight in Bleach Solution under strong light. Samples were rehydrated through a reverse methanol gradient (100% and 50%) and washed with PBST. Samples were then incubated with 3 μg/mL Proteinase K in a 37 °C shaker for 10 min, then fixed in 4% PFA at room temperature for 10 min. After repeated PBST washes, pre-hybridization at 56 °C for 2 h was performed. During hybridization, 1 μg/mL RNA probe was added, followed by 16 h hybridization at 56 °C. Post-hybridization washing was performed, followed by 2 h blocking with Blocking Solution and overnight incubation with Antibody Solution (anti-DIG-AP, 1:2000; Roche, Basel, Switzerland) at 4 °C. Maleic Acid Buffer with Tween 20 (MABT) washing was conducted six times at room temperature, followed by two short washes with AP Buffer. The color development solution was added and stored in the dark. When color development was complete, PBST was used to terminate the reaction. Background removal was achieved through sequential treatment with anhydrous ethanol and 50% ethanol. Finally, samples were washed with PBST, covered with glycerol gelatin, and photographed using a stereomicroscope.
2.6. Immunohistochemistry
The sample was washed with PBS multiple times, treated with 2% HCl for 5 min, and fixed with 4% PFA at 37 °C for 1 h. PBST was used for repeated washing, followed by methanol gradient dehydration (30%, 50%, 70%, and 100%). The sample was then dehydrated with 100% methanol at 20 °C for 2 h and bleached under strong light overnight. Rehydration was performed using methanol gradients (100%, 70%, 50%, and 30%), followed by PBST washing. The sample was permeabilized with 1% SDS for 2 h, washed with PBST, and then blocked with 5% BSA solution for 2 h. Anti-SYNORF1 (pan-neural marker to label synaptic neuropil) primary antibody (1:300; AB Company, Iowa City, IA, USA) was incubated at 4 °C overnight, followed by PBST washing and incubation with anti-mouse IgG/Cy3 secondary antibody (1:200; EarthOx, Millbrae, CA, USA) at 4 °C for 2 h. Finally, images were captured using a stereomicroscope.
2.7. RNA-Seq Analysis
Prior to control or ADCY9 RNAi treatment (5 × RNAi), planarians were subjected to 7 days of starvation. Total RNA was extracted from snap-frozen planarians, with three biological replicates prepared (three individuals per replicate). RNA quality was assessed using an Agilent 2100 Bioanalyzer, and RNA concentration was determined with a NanoDrop ND-2000 spectrophotometer. Only high-quality RNA samples meeting the criteria (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, and >1 μg total RNA) were used for transcriptome library construction. Bioinformatic analysis was carried out on the Majorbio Cloud platform (www.majorbio.com (accessed on 6 February 2025)). For the gene sets of interest, clustering analysis was performed based on relative expression levels, represented as log2-transformed fold changes between the two comparison groups. A distance matrix was generated using a corresponding distance algorithm to quantify pairwise similarities among genes. Iterative clustering was then applied to define gene subclusters according to their expression distance.
2.8. Statistical Analysis
qPCR data are expressed as the mean ± standard error of the mean (SEM). Statistical differences between groups were evaluated using Student’s t-test. Asterisks indicate statistically significant differences: * p < 0.05 and ** p < 0.01. All statistical analyses were performed using SPSS 16.0 software for Windows.
3. Results
3.1. Characterization of the Planarian ADCY9 Gene
To identify candidate genes associated with planarian head regeneration, we cloned the full-length cDNA of an ADCY9 homolog and designated it as ADCY9. The results revealed that ADCY9 was located at the base of the evolutionary tree, closer to the invertebrates, which conformed to the evolution order (red box) according the phylogenetic tree (Figure 1A). ADCY9’s homology was conserved in both invertebrates and vertebrates, according to protein sequence alignment (Figure 1B,C). Alignment of ADCY9 demonstrated above 30% identity with ADCY9s in vertebrates and 80.7% identity with ADCY9 in Schmidtea mediterranea (Figure 1B).
3.2. Spatiotemporal Expression Pattern of ADCY9 During Planarian Regeneration
To investigate the expression pattern of ADCY9 during planarian neural regeneration, qPCR and WISH were used to detect the expression characteristics of ADCY9 during planarian brain regeneration. As shown in Figure 2A, ADCY9 was broadly expressed in intact planarians, mainly distributed in the parenchyma and central nervous system, especially in the cephalic ganglia. The expression level of ADCY9 exhibited a significant time-dependent upregulation during planarian brain neural regeneration. Expression gradually increased in the early regeneration stage (1–3 dpa) and sharply elevated during the critical regeneration period (5–10 dpa), suggesting a vital role of ADCY9 in planarian cerebral neural regeneration (Figure 2B).
WISH revealed stage-specific spatial expression of ADCY9 during planarian brain regeneration: at 1 dpa, ADCY9 was concentrated in the blastema region (arrow); at 3 dpa, positive signals were detected in the brain primordium (arrow); as regeneration progressed, ADCY9 became significantly enriched in the newly formed cerebral ganglia (arrows) (Figure 2C). These findings indicate that ADCY9 displays a spatial progression of expression from the regeneration blastema to the brain ganglia during planarian brain regeneration, and its stage-specific enrichment may be synchronized with the reconstruction of neural structures.
Spatiotemporal expression of ADCY9 gene. (A) Expression profile of ADCY9 in intact planarians was determined by WISH. Scale bars: 500 μm. (B) qPCR was performed to examine ADCY9 transcript levels during planarian brain regeneration. (C) WISH of ADCY9 expression in planarian brain regeneration. Scale bars: 500 μm. * p < 0.05; ** p < 0.01.
3.3. Downregulation of ADCY9 Leads to Abnormal Neural Regeneration in Planarian Brain
To explore the functional role of ADCY9 during planarian brain regeneration, RNAi was performed to knockdown ADCY9 expression, and the structural integrity of cephalic ganglia as well as the branching complexity of lateral nerves were assessed. qPCR quantification revealed that ADCY9 transcript levels were significantly downregulated in regenerating fragments following decapitation, confirming the efficiency of RNAi-mediated silencing (Figure 3B). Fluorescence immunohistochemical analysis using an anti-SYNORF1 protein antibody revealed that the control groups exhibited significant neural regeneration efficiency during the critical regeneration period (5–10 dpa): planarian brain structures were gradually restored to completeness, and lateral nerve branches became increasingly complex. In contrast, compared to the control group, the ADCY9 (RNAi) group showed regional absence of newly formed cerebral nerve cords in the posterior region and a reduced number of regenerated collateral branches (Figure 3C). These results indicate that ADCY9 plays an important role in planarian cerebral neural regeneration.
3.4. Downregulation of ADCY9 Results in Reduced Neuronal Regeneration
Previous studies have identified multiple evolutionarily conserved neurotransmitters in planarians that are also present in vertebrates, along with their corresponding neural networks. Cholinergic, dopaminergic, serotonergic, and GABAergic neurons—all of which synthesize classical neurotransmitters—were found to be predominantly distributed in the nascent brain within 5–7 days post-amputation [22,23,24,25]. To verify whether ADCY9 influences the regeneration of distinct neuronal populations at 5, 7, and 10 dpa, neurotransmitter-synthesizing enzymes were employed as neuronal markers to count neuron numbers in decapitated planarians by WISH following ADCY9 knockdown. To further investigate the function of ADCY9 in planarian cerebral neural regeneration, WISH was used to detect the neuron markers (Chat (choline acetyltransferase), TH (tyrosine hydroxylase), GAD (glutamic acid decarboxylase), and TPH (tryptophan hydroxylase)) during planarian nerve regeneration upon ADCY9 RNAi. Morphological analysis showed that the proliferation rate of serotonergic neurons (TPH^+^) and cholinergic neurons (Chat^+^) was decreased, accompanied by decreased collateral nerve density and delayed neurogenesis at day 5–7 (Figure 4A,C,F,H). At the same time, the number of dopaminergic (TH^+^) and GABAergic (GAD^+^) neurons decreased significantly and showed spatial discrete distribution (Figure 4B,D,E,G). At 10 dpa, the number of neurons in the experimental group was significantly lower than that in the control group (Figure 4). These results suggest that ADCY9 plays a crucial role in the regeneration of planarian’s serotonergic, cholinergic, GABAergic and monoaminergic neurons and that the loss of ADCY9 function can cause developmental disorders in multiple neural cell types.
To explore the function of ADCY9 in neural stem cell homeostasis, we analyzed the expression of the neural stem cell (Ston2) in regenerating planarians, so as to evaluate alterations in the neural stem cell pool after ADCY9 knockdown. As illustrated in Figure 5A,B, Ston2 expression was significantly downregulated in planarians at 3 dpa following ADCY9 RNAi treatment (arrows). These results suggest that ADCY9 might regulate neural stem cells to maintain planarian cephalic ganglia regeneration.
The expression of different neurons in ADCY9 RNAi-treated regenerative planarians by WISH. (A) Expression analysis of cholinergic neurons (Chat+) by WISH during planarian regeneration at 5, 7 and 10 d. (B) Expression analysis of dopaminergic neurons(TH+) by WISH during planarian regeneration at 5 dpa, 7 dpa and 10 dpa. (C) qPCR analysis of Chat expression response to ADCY9 RNAi. n = 5. ** p < 0.01. (D) qPCR analysis of TH expression response to ADCY9 RNAi. n = 5. ** p < 0.01. (E) Expression analysis of GABAergic neurons (GAD+) by WISH during planarian regeneration at 5 dpa, 7 dpa and 10 dpa. (F) Expression analysis of serotonergic neurons (TPH+) by WISH during planarian regeneration at 5 dpa, 7 dpa and 10 dpa. (G) qPCR analysis of GAD expression response to ADCY9 RNAi. n = 5. ** p < 0.01. (H) qPCR analysis of TPH expression response to ADCY9 RNAi. n = 5. ** p < 0.01. Scale bars: 500 μm. The arrows refer to the positive signals.
Knockdown of ADCY9 causes brain primordia defective during planarian regeneration. (A) Expression analysis of Ston2 in regenerative planarians by WISH. Scale bars: 500 μm. (B) qPCR analysis of Ston2 expression response to ADCY9 RNAi. n = 5. ** p < 0.01.
3.5. Transcriptome Analysis After ADCY9 Gene Downregulation
To further elucidate the mechanism by which ADCY9 regulates planarian regeneration, RNA-seq was performed after RNAi-mediated ADCY9 knockdown. RNA-seq profiling revealed a total of 499 significantly differentially expressed genes (DEGs) following ADCY9 downregulation, with 235 genes upregulated and 264 genes downregulated (Figure 6A). KEGG pathway enrichment analysis of the upregulated DEGs indicated that the top 10 enriched pathways were closely related to the pathological mechanisms of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (Figure 6B). Notably, a set of genes, namely ATPase, NADH, Cytochrome, polyubiquitin, and Mitofusin-1, were screened out in this RNA-seq (Table S1). These findings provide a foundational framework for subsequent investigations into the regulatory mechanism of ADCY9 in planarian regeneration.
3.6. ADCY9 Regulates the Process of Nerve Regeneration Through Interacting Factors
To explore the downstream genetic interaction network of ADCY9, based on the planarian regeneration model, WISH and qPCR were used to examine the effect of ADCY9 and Mitofusin-1 double knockdown treatment on the expression of key neural markers (Ston2, Chat, and TH).
Morphological analysis of Ston2 showed that at 3 dpa (Figure 7A,B), the brain primordium of the ADCY9 and Mitofusin-1 double knockdown treatment group was significantly enlarged, and more complete nerve cords and abundant small nerve collateral branches were developed, indicating that co-knockdown of ADCY9 and Mitofusin-1 enhanced early neural structural remodeling.
At 10 dpa, this group showed systemic advantages in the regeneration of multiple neuronal subtypes. For cholinergic neuron (Chat^+^) regeneration, neurites were more complex and the structure of the new brain area was more complete (Figure 7C,D). For monoaminergic neuron regeneration, the structure of dopaminergic neurons (TH^+^) was more complete and showed broader signal distribution and higher expression levels, reflecting more comprehensive circuit reconstruction (Figure 7E,F). Overall, compared with the control group, the ADCY9 and Mitofusin-1 double knockdown treatment group showed more comprehensive and efficient circuit reconstruction ability in multiple dimensions of the neural structure.
ADCY9 regulates planarian cephalic ganglia regeneration through Mitofusin-1. (A,B) Ston2 expression in 3 dpa regenerating planarians subjected to ADCY9 + Mitofusin-1 double RNAi, analyzed by WISH and qPCR. Scale bars: 500 μm. n = 5. (C,D) Chat expression at 10 dpa in regenerating planarians subjected to ADCY9 + Mitofusin-1 double RNAi, analyzed by WISH and qPCR. Scale bars: 500 μm. n = 5. (E,F) TH expression at 10 dpa in regenerating planarians subjected to ADCY9 + Mitofusin-1 double RNAi, analyzed by WISH and qPCR. Scale bars: 500 μm. n = 5. NS, not significant.
4. Discussion
Planarians represent an optimal in vivo model for investigating nervous system regeneration. During this process, neural stem cells need to maintain appropriate proliferation while their newly differentiated progeny is properly integrated into patterned tissues. In this study, analysis of the ADCY9 expression profile revealed temporally upregulated expression from the early to the critical regeneration phase, along with a spatially progressive expression pattern from the blastema to the cephalic ganglia, suggesting that ADCY9 may act as a key regulator of neural progenitor cell migration and differentiation during regeneration. This expression pattern was consistent with the documented functions of adenylate cyclase family members in the nervous systems of various model organisms, further supporting its conserved role in cell signaling and morphogenesis [26,27,28,29,30,31].
In terms of function, based on the planarian regeneration model, we found that the loss of ADCY9 not only leads to abnormal development of the nerve cord structure but also causes coordinated defects in the number and spatial distribution of multiple neuronal subtypes. This indicates that ADCY9 systemically promotes neural network reconstruction by coordinating the differentiation programs of heterogeneous neurons. This phenotypic feature aligns closely with the functional mechanisms of ADCY9 in the mammalian nervous system. In mammals, ADCY9 acts as a key node for Ca^2+^/cAMP signal integration. By regulating the dynamic balance of cAMP signaling, it coordinates downstream networks related to cell survival, inflammation, and structural plasticity, thereby participating in synaptic plasticity and cognitive processes, as well as the regulation of neuroinflammation and injury repair [17,32]. Thus, ADCY9 may, through conserved pathways, coordinate the spatiotemporal differentiation of multiple neuronal types, thereby driving the overall reconstruction of neural networks.
RNA-seq analysis further revealed that ADCY9 knockdown caused 499 differentially expressed genes, among which KEGG enriched pathways were significantly associated with neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases. This finding is consistent with previous studies showing that ADCY9 is specifically highly expressed in advanced cognitive brain regions such as the mammalian hippocampus and is regulated by the Ca^2+^/calcineurin signaling axis, playing a key role in synaptic plasticity, memory consolidation, and the maintenance of neural circuit homeostasis [32,33]. Combined with the phenotypic evidence from this study, dysregulation of ADCY9 may disrupt this conserved signaling network, affecting not only structural reconstruction during neural development and regeneration but also neuronal survival, differentiation, and synaptic integrity, thereby suggesting a potential association with the pathogenesis of neurodegenerative diseases. This provides new clues for understanding the unified regulatory role of ADCY9 in physiological and pathological neural plasticity.
The results of the double interference experiment indicated that ADCY9, as a key positive regulator of planarian nerve regeneration, may regulate the regeneration process by interacting with the downstream inhibitory element Mitofusin-1. Specifically, the genetic interaction between ADCY9 and Mitofusin-1 was characterized by a linear regulatory pattern. This model predicts that ADCY9 acts as an upstream regulator to mediate its pro-regenerative function by negatively regulating the downstream repressor Mitofusin-1. The combined interference of ADCY9 and Mitofusin-1 can relieve the inhibitory effect of Mitofusin-1 on the regeneration process to a certain extent, thus supporting the continuous and efficient regeneration process. This finding is consistent with conclusions drawn from studies on embryonic-derived human and mouse neural stem cells, which indicate that, as a key fusion protein on the mitochondrial outer membrane, Mitofusin-1-mediated mitochondrial fusion enhances the self-renewal of neural stem cells, while its functional inhibition and mitochondrial network fragmentation promote neuronal differentiation and maturation. This mechanism corresponds to the phenotypic characteristics observed in experiments and aligns with research conclusions on mitochondrial dynamics regulating neurogenesis in mammalian models [34,35,36].
In summary, this study systematically analyzed the function of ADCY9 in planarian nerve regeneration. By directing the proliferation and spatial localization of neural precursor cells, ADCY9 orchestrates multiple neuronal differentiation and neural remodeling processes. The dual interference experiment further showed that Mitofusin-1 was involved in a downstream regulatory network, which regulated the regeneration intensity in a hierarchical manner. This study provided a new theoretical framework for further elucidating the molecular regulatory mechanism of nerve regeneration.
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