Deciphering the Akt1-HuD interaction in HuD-mediated neuronal differentiation.

The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.

RNA binding protein HuD mediates the crosstalk between ß cells and islet endothelial cells by the regulation of Endostatin and Serpin E1 expression.

RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma ßTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3'-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3'UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of ßTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between ß cells and islet endothelial cells, and the results showed that HuD downregulation in ßTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between ß cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.

RNA-binding protein ELAVL4/HuD ameliorates Alzheimer's disease-related molecular changes in human iPSC-derived neurons.

The RNA binding protein ELAVL4/HuD regulates the translation and splicing of multiple Alzheimer's disease (AD) candidate genes. We generated ELAVL4 knockout (KO) human induced pluripotent stem cell-derived neurons to study the effect that ELAVL4 has on AD-related cellular phenotypes. ELAVL4 KO significantly increased the levels of specific APP isoforms and intracellular phosphorylated tau, molecular changes that are related to the pathological hallmarks of AD. Overexpression of ELAVL4 in wild-type neurons and rescue experiments in ELAVL4 KO cells showed opposite effects and also led to a reduction of the extracellular amyloid-beta (Aß)42/40 ratio. All these observations were made in familial AD (fAD) and fAD-corrected neurons. To gain insight into the molecular cascades involved in neuronal ELAVL4 signaling, we conducted pathway and upstream regulator analyses of transcriptomic and proteomic data from the generated neurons. These analyses revealed that ELAVL4 affects multiple biological pathways linked to AD, including those involved in synaptic function, as well as gene expression downstream of APP and tau signaling. The analyses also suggest that ELAVL4 expression is regulated by insulin receptor-FOXO1 signaling in neurons. Taken together, ELAVL4 expression ameliorates AD-related molecular changes in neurons and affects multiple synaptic pathways, making it a promising target for novel drug development.

Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype.

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic ß-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3'-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.

ALS-related FUS mutations alter axon growth in motoneurons and affect HuD/ELAVL4 and FMRP activity.

Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3'UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

Identification of Compounds Targeting HuD. Another Brick in the Wall of Neurodegenerative Disease Treatment.

ELAV-like (ELAVL) RNA-binding proteins play a pivotal role in post-transcriptional processes, and their dysregulation is involved in several pathologies. This work was focused on HuD (ELAVL4), which is specifically expressed in nervous tissues, and involved in differentiation and synaptic plasticity mechanisms. HuD represents a new, albeit unexplored, candidate target for the treatment of several relevant neurodegenerative diseases. The aim of this pioneering work was the identification of new molecules able to recognize and bind HuD, thus interfering with its activity. We combined virtual screening, molecular dynamics (MD), and STD-NMR techniques. Starting from around 51 000 compounds, four promising hits eventually provided experimental evidence of their ability to bind HuD. Among the selected best hits, folic acid was found to be the most interesting one, being able to well recognize the HuD binding site. Our results provide a basis for the identification of new HuD interfering compounds which may be useful against neurodegenerative syndromes.

The RNA-Binding Protein HuD Regulates Alternative Splicing and Alternative Polyadenylation in the Mouse Neocortex.

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.

HuB and HuD repress telomerase activity by dissociating HuR from TERC.

The ubiquitous RNA-binding protein HuR (ELAVL1) promotes telomerase activity by associating with the telomerase noncoding RNA TERC. However, the role of the neural-specific members HuB, HuC, and HuD (ELAVL2-4) in telomerase activity is unknown. Here, we report that HuB and HuD, but not HuC, repress telomerase activity in human neuroblastoma cells. By associating with AU-rich sequences in TERC, HuB and HuD repressed the assembly of the TERT-TERC core complex. Furthermore, HuB and HuD competed with HuR for binding to TERC and antagonized the function of HuR that was previously shown to enhance telomerase activity to promote cell growth. Our findings reveal a novel mechanism controlling telomerase activity in human neuroblastoma cells that involves a competition between HuR and the related, neural-specific proteins HuB and HuD.

IGFBPs mediate IGF-1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids.

Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.

HuD regulates SOD1 expression during oxidative stress in differentiated neuroblastoma cells and sporadic ALS motor cortex.

The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.

The RNA-binding protein, HuD regulates proglucagon biosynthesis in pancreatic α cells.

Glucagon is a peptide hormone generated by pancreatic α cells. It is the counterpart of insulin and plays an essential role in the regulation of blood glucose level. Therefore, a tight regulation of glucagon levels is pivotal to maintain homeostasis of blood glucose. However, little is known about the mechanisms regulating glucagon biosynthesis. In this study, we demonstrate that the RNA-binding protein HuD regulates glucagon expression in pancreatic α cells. HuD was found in α cells from mouse pancreatic islet and mouse glucagonoma αTC1 cell line. Ribonucleoprotein immunoprecipitation analysis, followed by RT-qPCR showed the association of HuD with glucagon mRNA. Knockdown of HuD resulted in a reduction in both proglucagon expression and cellular glucagon level by decreasing its de novo synthesis. Reporter analysis using the EGFP reporter containing 3' untranslated region (3'UTR) of glucagon mRNA showed that HuD regulates proglucagon expression via its 3'UTR. In addition, the relative level of glucagon in the islets and plasma was lower in HuD knockout (KO) mice compared to age-matched control mice. Taken together, these results suggest that HuD is a novel factor regulating the biosynthesis of proglucagon in pancreatic α cells.

RNA binding protein HuD and microRNA-203a cooperatively regulate insulinoma-associated 1 mRNA.

RNA binding protein HuD regulates translation and turnover of target mRNAs, thereby affecting gene expression at the posttranscriptional level in mainly neuronal as well as pancreatic ß-cells. Here, we identified insulinoma-associated 1 (INSM1), an essential factor governing differentiation and proliferation of neuroendocrine cells, as a novel target of HuD and demonstrated the regulatory mechanism of INSM1 expression by HuD. HuD bound to 3'untranslated region (3'UTR) of Insm1 mRNA and negatively regulated its expression; knockdown of HuD increased INSM1 expression, while HuD overexpression repressed it by destabilizing its mRNA. In addition, we further demonstrated that HuD enhanced reduction of INSM1 by miR-203a, a novel miRNA targeting Insm1 mRNA 3'UTR. These results suggest that HuD and miR-203a cooperatively regulate INSM1 expression and it provides a novel regulatory mechanism of INSM1 expression by HuD and miR-203a.

RNA binding protein HuD contributes to ß-cell dysfunction by impairing mitochondria dynamics.

Imbalanced mitochondrial dynamics in pancreatic ß-cells contributes to ß-cell dysfunction in diabetes; however, the molecular mechanisms underlying mitochondrial dynamics in the pathology of diabetes are not fully elucidated. We previously reported the reduction of RNA binding protein HuD in pancreatic ß-cells of diabetes. Herein, we demonstrate that HuD plays a novel role in the regulation of mitochondrial dynamics by promoting mitochondrial fusion. We show enhanced mitochondrial fragmentation in the pancreas of db/db mice and HuD KO mice. Downregulation of HuD increases the number of cells with fragmented mitochondria and reduces the mitochondrial activity determined by mitochondrial membrane potential and ATP production in mouse insulinoma ßTC6 cells. HuD binds to 3'-untraslated region of mitofusin 2 (Mfn2) mRNA and positively regulates its expression. Ectopic expression of Mfn2 in ßTC6 cells stably expressing short hairpin RNA against HuD (shHuD) restores HuD-mediated mitochondrial dysfunction. Taken together, our results suggest that HuD regulates mitochondrial dynamics by regulating Mfn2 level and its reduced expression leads to mitochondrial dysfunction in pancreatic ß-cells.

Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3' UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.

RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice.

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene.

MicroRNAs regulate post-transcriptional gene expression via either translational repression or mRNA degradation. They have important roles in both viral infection and host anti-infection processes. We discovered that the miR-375 is significantly upregulated in Newcastle disease virus (NDV)-infected chicken embryonic visceral tissues using a small RNA sequencing approach. Further research revealed that the overexpression of miR-375 markedly decreases the replication of the velogenic NDV F48E9 and the lentogenic NDV La Sota by targeting the M gene of NDV in DF-1 cells. Interestingly, miR-375 has another target, ELAVL4, which regulates chicken fibrocyte cell cycle progression and decreases NDV proliferation. In addition, miR-375 can influence bystander cells by its secretion in culture medium. Our results indicated that miR-375 is an inhibitor of NDV, but can also enhance NDV growth by reducing the expression of its target ELAVL4. These results emphasize the complex roles of microRNAs in the regulation of viral infections.

Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD.

The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 isoform expression. We show that HuR and HuD inhibit activity at the intron 9 polyadenylation site. When co-expressed with the KCNH2 gene, HuR and HuD increased levels of the Kv11.1a isoform and decreased the Kv11.1a-USO isoform in the RNase protection assays and immunoblot analyses. In patch clamp experiments, HuR and HuD significantly increased the Kv11.1 current. siRNA-mediated knockdown of HuR protein decreased levels of the Kv11.1a isoform and increased those of the Kv11.1a-USO isoform. Our findings suggest that the relative expression levels of Kv11.1 C-terminal isoforms are regulated by the RNA-binding HuR and HuD proteins.

Ndr kinases regulate retinal interneuron proliferation and homeostasis.

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.