Stub1 promotes degradation of the activated Diaph3: A negative feedback regulatory mechanism of the actin nucleator.

The formin protein Diaph3 is an actin nucleator that regulates numerous cytoskeleton-dependent cellular processes through the activation of actin polymerization. Expression and activity of Diaph3 is tightly regulated: lack of Diaph3 results in developmental defects and embryonic lethality in mice, while overexpression of Diaph3 causes auditory neuropathy. It is known that Diaph3 homophilic interactions include the intramolecular interaction of its Dia-inhibitory domain (DID)-diaphanous autoregulatory domain (DAD) domains and the intermolecular interactions of DD-DD domains or FH2-FH2 domains. However, the physiological significance of these interactions in Diaph3 protein stability and activity is not fully understood. In this study, we show that FH2-FH2 interaction promotes Diaph3 activity, while DID-DAD and DD-DD interactions inhibit Diaph3 activity through distinct mechanisms. DID-DAD interaction is responsible for the autoinhibition of Diaph3 protein, which is disrupted by binding of Rho GTPases. Interestingly, we find that DID-DAD interaction stabilizes the expression of each DID or DAD domain against proteasomal-mediated degradation. Disruption of DID-DAD interaction by RhoA binding or M1041A mutation causes increased Diaph3 activity and accelerated degradation of the activated Diaph3 protein. Further, the activated Diaph3 is ubiquitinated at K1142/1143/1144 lysine residues by the E3 ligase Stub1. Expression of Stub1 is causally related to the stability and activity of Diaph3. Knockdown of Stub1 in mouse cochlea results in hair cell stereocilia defects, neuronal degeneration, and hearing loss, resembling the phenotypes of mice overexpressing Diaph3. Thus, our study reports a novel regulatory mechanism of Diaph3 protein expression and activity whereby the active but not inactive Diaph3 is readily degraded to prevent excessive actin polymerization.

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel.

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

Thyroid hormone controls the timing of cochlear ribbon synapse maturation.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution.

Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and injured tissue nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord surgery. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+ , S100a4+ , Gpr84+ , C3+ /G0s2+ , GFAP+ /Tm4sf1+ , and Gss+ /Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphologically and physiologically distinct astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understand the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Spontaneous regeneration of cochlear supporting cells after neonatal ablation ensures hearing in the adult mouse.

Supporting cells in the cochlea play critical roles in the development, maintenance, and function of sensory hair cells and auditory neurons. Although the loss of hair cells or auditory neurons results in sensorineural hearing loss, the consequence of supporting cell loss on auditory function is largely unknown. In this study, we specifically ablated inner border cells (IBCs) and inner phalangeal cells (IPhCs), the two types of supporting cells surrounding inner hair cells (IHCs) in mice in vivo. We demonstrate that the organ of Corti has the intrinsic capacity to replenish IBCs/IPhCs effectively during early postnatal development. Repopulation depends on the presence of hair cells and cells within the greater epithelial ridge and is independent of cell proliferation. This plastic response in the neonatal cochlea preserves neuronal survival, afferent innervation, and hearing sensitivity in adult mice. In contrast, the capacity for IBC/IPhC regeneration is lost in the mature organ of Corti, and consequently IHC survival and hearing sensitivity are impaired significantly, demonstrating that there is a critical period for the regeneration of cochlear supporting cells. Our findings indicate that the quiescent neonatal organ of Corti can replenish specific supporting cells completely after loss in vivo to guarantee mature hearing function.

Inner ear supporting cells: rethinking the silent majority.

Sensory epithelia of the inner ear contain two major cell types: hair cells and supporting cells. It has been clear for a long time that hair cells play critical roles in mechanoreception and synaptic transmission. In contrast, until recently the more abundant supporting cells were viewed as serving primarily structural and homeostatic functions. In this review, we discuss the growing information about the roles that supporting cells play in the development, function and maintenance of the inner ear, their activities in pathological states, their potential for hair cell regeneration, and the mechanisms underlying these processes.

Metabolic Profiling of Cochlear Organoids Identifies α-Ketoglutarate and NAD+ as Limiting Factors for Hair Cell Reprogramming.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.

A human-specific cytotoxic neopeptide generated by the deafness gene Cingulin.

Accumulation of mutant proteins in cells can induce proteinopathies and cause functional damage to organs. Recently, the Cingulin (CGN) protein has been shown to maintain the morphology of cuticular plates of inner ear hair cells and a frameshift mutation in CGN causes autosomal dominant non-syndromic hearing loss. Here, we find that the mutant CGN proteins form insoluble aggregates which accumulate intracellularly and lead to cell death. Expression of the mutant CGN in the inner ear results in severe hair cell death and hearing loss in mice, resembling the auditory phenotype in human patients. Interestingly, a human-specific residue (V1112) in the neopeptide generated by the frameshift mutation is critical for the aggregation and cytotoxicity of the mutant human CGN. Moreover, the expression of heat shock factor 1 (HSF1) decreases the accumulation of insoluble mutant CGN aggregates and rescues cell death. In summary, these findings identify mutant-specific toxic polypeptides as a disease-causing mechanism of the deafness mutation in CGN, which can be targeted by the expression of the cell chaperone response regulator HSF1.

Macrophages and pulmonary fibrosis: a bibliometric and visual analysis of publications from 1990 to 2023.

Background: The role of macrophages in the symptomatic and structural progression of pulmonary fibrosis (PF) has garnered significant scholarly attention in recent years. This study employs a bibliometric approach to examine the present research status and areas of focus regarding the correlation between macrophages and PF, aiming to provide a comprehensive understanding of their relationship. Methodology: The present study employed VOSviewer, CiteSpace, and Microsoft Excel software to visualize and analyze various aspects such as countries, institutions, authors, journals, co-cited literature, keywords, related genes, and diseases. These analyses were conducted using the Web of Science core collection database. Results: A comprehensive collection of 3,479 records pertaining to macrophages and PF from the period of 1990 to 2023 was obtained. Over the years, there has been a consistent increase in research literature on this topic. Notably, the United States and China exhibited the highest level of collaboration in this field. Through careful analysis, the institutions, authors, and prominent journals that hold significant influence within this particular field have been identified as having the highest publication output. The pertinent research primarily concentrates on the domains of Biology and Medicine. The prevailing keywords encompass pulmonary fibrosis, acute lung injury, idiopathic pulmonary fibrosis, and others. Notably, TGFß1, TNF, and CXCL8 emerge as the most frequently studied targets, primarily associated with signaling pathways such as cytokine-cytokine receptor interaction. Additionally, cluster analysis of related diseases reveals their interconnectedness with ailments such as cancer. Conclusion: The present study employed bibliometric methods to investigate the knowledge structure and developmental trends in the realm of macrophage and PF research. The findings shed light on the introduction and research hotspots that facilitate a more comprehensive understanding of macrophages and PF.

TMEM63B channel is the osmosensor required for thirst drive of interoceptive neurons.

Thirst plays a vital role in the regulation of body fluid homeostasis and if deregulated can be life-threatening. Interoceptive neurons in the subfornical organ (SFO) are intrinsically osmosensitive and their activation by hyperosmolarity is necessary and sufficient for generating thirst. However, the primary molecules sensing systemic osmolarity in these neurons remain elusive. Here we show that the mechanosensitive TMEM63B cation channel is the osmosensor required for the interoceptive neurons to drive thirst. TMEM63B channel is highly expressed in the excitatory SFO thirst neurons. TMEM63B deletion in these neurons impaired hyperosmolarity-induced drinking behavior, while re-expressing TMEM63B in SFO restored water appetite in TMEM63B-deficient mice. Remarkably, hyperosmolarity activates TMEM63B channels, leading to depolarization and increased firing rate of the interoceptive neurons, which drives drinking behavior. Furthermore, TMEM63B deletion did not affect sensitivities of the SFO neurons to angiotensin II or hypoosmolarity, suggesting that TMEM63B plays a specialized role in detecting hyperosmolarity in SFO neurons. Thus, our results reveal a critical osmosensor molecule for the generation of thirst perception.

Cingulin regulates hair cell cuticular plate morphology and is required for hearing in human and mouse.

Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.

High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers.

MicroRNAs are small noncoding RNAs that serve as important regulators of eukaryotic gene expression and are emerging as novel diagnostic and therapeutic targets for human diseases. Robust and reliable detection of miRNAs is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Existing methods for miRNA quantification rely on fluorescent probes for optimal specificity. In this study, we developed a high-performance real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay that allows specific and rapid detection of mature miRNAs using a fast thermocycling profile (10 sec per cycle). This assay exhibited a wide dynamic range (>7 logs) and was capable of detecting miRNAs from as little as 1 pg of the total RNA or as few as 10 cells. The use of modified reverse-transcription oligonucleotides with a secondary structure and hemi-nested reverse PCR primers allowed excellent discrimination of mature miRNAs from their precursors and highly homologous family members using SYBR Green I. Using a novel approach involving uracil-DNA glycosylase treatment, we showed that carryover of the reverse transcription oligonucleotide to the PCR can be successfully eliminated and discrimination between miRNA homologs could be further enhanced. These assays were further extended for multiplexed detection of miRNAs directly from cell lysates without laborious total RNA isolation. With the robust performance of these assays, we identified several miRNAs that were regulated by glial cell-line-derived neurotrophic factor in human glioblastoma cells. In summary, this method could provide a useful tool for rapid, robust, and cost-effective quantification of existing and novel miRNAs.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses.

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8-P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

High-throughput screening on cochlear organoids identifies VEGFR-MEK-TGFB1 signaling promoting hair cell reprogramming.

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.

Cochlear Sox2+ Glial Cells Are Potent Progenitors for Spiral Ganglion Neuron Reprogramming Induced by Small Molecules.

In the mammalian cochlea, spiral ganglion neurons (SGNs) relay the acoustic information to the central auditory circuits. Degeneration of SGNs is a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear glial cells are able to form spheres and differentiate into neurons in vitro. However, the identity of these progenitor cells is elusive, and it is unclear how to differentiate these cells toward functional SGNs. In this study, we found that Sox2+ subpopulation of cochlear glial cells preserves high potency of neuronal differentiation. Interestingly, Sox2 expression was downregulated during neuronal differentiation and Sox2 overexpression paradoxically inhibited neuronal differentiation. Our data suggest that Sox2+ glial cells are potent SGN progenitor cells, a phenotype independent of Sox2 expression. Furthermore, we identified a combination of small molecules that not only promoted neuronal differentiation of Sox2- glial cells, but also removed glial cell identity and promoted the maturation of the induced neurons (iNs) toward SGN fate. In summary, we identified Sox2+ glial subpopulation with high neuronal potency and small molecules inducing neuronal differentiation toward SGNs.

Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells.

BACKGROUND: Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs), beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions. RESULTS: Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder. CONCLUSIONS: The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes during neuronal differentiation of PC12 cells, regardless of the type of differentiation conditions. The combination of these two novel reference genes, but not the commonly used HKG, GAPDH, allows robust and accurate normalization of differentially expressed genes during PC12 differentiation.

A specific isoform of glial cell line-derived neurotrophic factor family receptor alpha 1 regulates RhoA expression and glioma cell migration.

Malignant gliomas are highly invasive neuroepithelial tumors where the tendency to invade and migrate away from the primary tumor mass is thought to be a leading cause of tumor recurrence and treatment failures. Autocrine signals produced by secreted factors that signal through receptors on the tumor are known to contribute to the invasiveness. Glial cell line-derived neurotrophic factor and GDNF family receptor alpha 1 (GFRα1) are over-expressed in human gliomas. We have previously reported that human gliomas express high levels of GFRα1b, an alternatively spliced isoform of GFRα1. However, the functional significance of GFRα1b in glioma behaviors is currently unknown. In this study, we have designed isoform-specific small-interfering RNA to knockdown the highly homologous GFRα1a or GFRα1b isoform efficiently in malignant C6 glioma cells. Unexpectedly, the knockdown of GFRα1b but not GFRα1a induced cell elongation and inhibited C6 cell migration and invasion in vitro. In addition, GFRα1b was found to regulate the expression of RhoA small GTPase, which was required for migration of C6 cells. The decreases in RhoA expression and cell migration after GFRα1b knockdown were attenuated by small-interfering RNA -resistant GFRα1b but not GFRα1a, further demonstrating the specific role of GFRα1b in glioma migration. Interestingly, the knockdown of NCAM but not receptor tyrosine kinase Ret resulted in the reduction of RhoA expression and C6 cell migration. Taken together, these unanticipated results indicate that GFRα1b is involved in glioma migration through glial cell line-derived neurotrophic factor -GFRα1b-NCAM signaling complex and modulation of RhoA expression.

Identification and validation of reference genes for expression studies in a rat model of neuropathic pain.

Neuropathic pain is triggered by damage to or as a result of the dysfunction of the somatosensory nervous system. Gene expression profiling using DNA microarray and real-time PCR have emerged as powerful tools for the elucidation of pain-specific pathways and identification of candidate biomarkers and therapeutic targets. Proper normalization of the gene expression data with stable reference genes is a prerequisite to obtaining accurate gene expression changes. We have evaluated the stability of six candidate reference genes which include three commonly used housekeeping genes (ACTB, GAPDH and HMBS) and three ribosomal protein genes (RPL3, RPL19 and RPL29) using real-time PCR in a rat model of neuropathic pain. Unexpectedly, ACTB but not GAPDH was stably expressed. In addition, we have identified RPL29 and RPL3 as novel reference genes. Normalization of expression data using GAPDH or HMBS led to overestimation of transcriptional changes. Using RPL29/RPL3/ACTB as reference genes, a number of transcripts were found to be specifically and significantly regulated in injured dorsal root ganglia. These genes may contribute to the development of neuropathic pain pathology and may serve as candidate biomarkers for potential diagnosis.

GDNF-induced cell signaling and neurite outgrowths are differentially mediated by GFRalpha1 isoforms.

Glial cell line-derived neurotrophic factor (GDNF) transduces signal and promotes neurite outgrowths in diverse neurons through the interactions of GDNF family receptor alpha 1 (GFRalpha1) and other co-receptors including Ret receptor tyrosine kinase and NCAM. GFRalpha1 is alternatively spliced into two isoforms, GFRalpha1a and GFRalpha1b, with five amino acids difference. In this study, we found that both GFRalpha1a and GFRalpha1b were expressed in various human tissues. Interestingly, when stimulated with GDNF, GFRalpha1a but not GFRalpha1b promoted neurite outgrowth in neuroblastoma cells through the activations of ERK1/2, Rac1 and Cdc42. Remarkably, in cells co-expressing GFRalpha1a and GFRalpha1b, GDNF inhibited neurite outgrowths. The inhibitory activity of GFRalpha1b was dependent on RhoA and ROCK activation. Furthermore, GFRalpha1b but not GFRalpha1a activated Rho and various ROCK downstream effectors LIMK1/2, cofilin and MLC2. This study demonstrates the hitherto unrecognized roles of GFRalpha1 isoforms in the activation of distinct signaling pathways and in neurite outgrowths.