Growth Of Hippocampal Neurons Research Articles

Calmodulin Triggers Activity-Dependent rRNA Biogenesis via Interaction with DDX21.

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

Gamma secretase activity modulates BMP-7-induced dendritic growth in primary rat sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

Issue Information

Journal of NeurochemistryVolume 165, Issue 6 p. 757-758 ISSUE INFORMATIONFree Access Issue Information First published: 22 June 2023 https://doi.org/10.1111/jnc.15634 Read the full article: ‘Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory’ by R. Martínez-Mármol, Y. Chai, J. N. Conroy, Z. Khan, S.-M. Hong, S. B. Kim, R. S. Gormal, D. H. Lee, J. K. Lee, E. J. Coulson, M. K. Lee, S. Y. Kim, and F. A. Meunier (J. Neurochem. vol. 165 (6), pp. 791–808) on doi:10.1111/jnc.15767 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Front cover The traditional medicinal mushroom Hericium erinaceus, also known as lion’s mane mushroom has bioactive compounds with neurotrophic activity. Here, we purified and identified biologically active molecules isolated from H. erinaceus, based on their ability to promote neurite extensive outgrowth in hippocampal neurons. We found that N-de phenylethyl isohericerin (NDPIH) and its hydrophobic derivative hericene A, were highly potent in inducing neurite outgrowth and branching in cultured hippocampal neurons. In addition, mice feed with hericene A showed both increased BDNF signaling and enhanced hippocampal memory performance. Biochemical analysis revealed that the aforementioned neurotrophic effects are independent but convergent to BDNF/TrkB pathway. Our results reveal potent bioactive compounds derived from H. erinaceus. These molecules are very attractive compounds for their neurotrophic properties. Our findings open the door to identifying novel mushroom-derived compounds as an strategy for the development of sustainable therapeutical interventions to revert the cognitive decline associated with ageing and neurodegenerative diseases. Image content Confocal image of a giant growth cone from an hippocampal neuron elicited by NDPIH. Immunocytochemistry against tubulin (mouse anti-βIII tubulin primary antibody, and Alexa FluorTM 488-goat-anti-mouse secondary antibody, green) and actin (Alexa-FluorTM 647-phalloidin, magenta). Read the full article ‘Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory’ by R. Martínez-Mármol, Y. Chai, J. N. Conroy, Z. Khan, S.-M. Hong, S. B. Kim, R. S. Gormal, D. H. Lee, J. K. Lee, E. J. Coulson, M. K. Lee, S. Y. Kim, and F. A. Meunier (J. Neurochem. vol. 165 (6), pp. 791–808) on doi:10.1111/jnc.15767 Volume165, Issue6June 2023Pages 757-758 RelatedInformation

Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory.

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

Rab3A interacts with spastin to regulate neurite outgrowth in hippocampal neurons

Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.

Treadmill training improves cognitive function by increasing IGF2 targeted downregulation of miRNA-483.

Optimal exercise can promote the development of cognitive functions. Nevertheless, mechanisms that elicit these positive effects of exercise still need to be elucidated. Insulin-like growth factor 2 (IGF2) is known to act as a potent enhancer of memory and cognitive functions, whereas the mechanism by which IGF2 regulates cognitive functions in terms of moderate treadmill exercise remains largely vague. In the study, rats were subjected to low-, moderate-, and high-intensity treadmill training for 6 weeks. Then, the Morris water maze test was used to investigate spatial learning and memory ability in rats subjected to treadmill exercises of different intensities. Subsequently, gene chip and bioinformatics analyses were used to explore IGF2 and predict target microRNAs (miRNAs). Quantitative real-time polymerase chain reaction, western blot, and immunofluorescence analysis were performed to detect the levels of IGF2. Furthermore, IGF2-small interfering RNA, the miRNA-483-mimic, and the miRNA-483-inhibitor were transfected to determine the role of IGF2 and miRNA-483 in the growth of hippocampal neurons. The results of the Morris water maze test showed that moderate-intensity treadmill training enhanced cognitive functions; meanwhile, the expression of IGF2 was significantly upregulated in the hippocampus after moderate-intensity treadmill exercise. From databases, miRNA-483 was screened and predicted as the target gene of IGF2. Moreover, silencing IGF2 inhibited neurite growth in the hippocampus of rats, the miRNA-483-inhibitor ameliorated silencing IGF2 induced impairment of hippocampal neurons. These findings suggested that treadmill training could enhance cognitive functions, wherein the underlying mechanism involved an increase in the expression of IGF2 and downregulation of miRNA-483.

Regulatory effects of gradient microtopographies on synapse formation and neurite growth in hippocampal neurons

Among approaches aiming toward functional nervous system restoration, those implementing microfabrication techniques allow the manufacture of platforms with distinct geometry where neurons can develop and be guided to form patterned connections in vitro. The interplay between neuronal development and the microenvironment, shaped by the physical limitations, remains largely unknown. Therefore, it is crucial to have an efficient way to quantify neuronal morphological changes induced by physical or contact guidance of the microenvironment. In this study, we first devise and assess a method to prepare anisotropic, gradient poly(dimethylsiloxane) micro-ridge/groove arrays featuring variable local pattern width. We then demonstrate the ability of this single substrate to simultaneously profile the morphologcial and synaptic connectivity changes of primary cultured hippocampal neurons reacting to variable physical conditons, throughout neurodevelopment, in vitro. The gradient microtopography enhanced adhesion within microgrooves, increasing soma density with decreasing pattern width. Decreasing pattern width also reduced dendritic arborization and increased preferential axon growth. Finally, decreasing pattern geometry inhibited presynaptic puncta architecture. Collectively, a method to examine structural development and connectivity in response to physical stimuli is established, and potentially provides insight into microfabricated geometries which promote neural regeneration and repair.

MiR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro.

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3′UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.

Regulation of dendrite growth by Cdc42 effector protein‑4 in hippocampal neurons in vitro.

Cell division control protein 42 homolog (Cdc42), one of the most characteristic members of the Rho protein family, is required for multiple aspects of dendritic morphogenesis. However, the proteins mediating the regulatory effects of Cdc42 activity on neuronal morphology are largely unknown. Cdc42 effector protein-4 (CEP4) was identified to be a binding partner of Rho GTPase 4 and is ubiquitously expressed in all adult tissues. However, the physiological function of CEP4 in neurons is unknown. In the present study, immunofluorescence and western blot analysis were conducted, revealing that CEP4 is highly expressed in the brain, and that the expression of CEP4 is gradually increased during neurodevelopment. Knockdown of CEP4 with short hairpin RNA suppressed dendrite growth, whereas overexpression of wild-type CEP4 promoted dendrite growth in primary isolated mouse hippocampal neurons. Collectively, these results indicated an important role for CEP4 in dendrite growth in hippocampal neurons.

Knock-Down of CD24 in Astrocytes Aggravates Oxyhemoglobin-Induced Hippocampal Neuron Impairment.

Subarachnoid hemorrhage (SAH), as one of the most severe hemorrhagic strokes, is closely related to neuronal damage. Neurogenesis is a promising therapy, however, reliable targets are currently lacking. Increasing evidence has indicated that CD24 is associated with the growth of hippocampal neurons and the regulation of neural stem/precursor cell proliferation. To investigate the potential effect of CD24 in astrocytes on neuron growth in the hippocampus, we used a Transwell co-culture system of hippocampal astrocytes and neurons, and oxyhemoglobin (OxyHb) was added to the culture medium to mimic SAH in vitro. A specific lentivirus was used to knock down CD24 expression in astrocytes, which was verified by western blot, quantitative real-time polymerase chain reaction, and immunofluorescent staining. Astrocyte activation, neurite elongation, neuronal apoptosis, and cell viability were also assessed. We first determined the augmented expression level of CD24 in hippocampal astrocytes after SAH. A similar result was observed in cultured astrocytes exposed to OxyHb, and a corresponding change in SHP2/ERK was also noticed. CD24 in astrocytes was then downregulated by the lentivirus, which led to the impairment of axons and dendrites on the co-cultured neurons. Aggravated neuronal apoptosis was induced by the CD24 downregulation in astrocytes, which might be a result of a lower level of brain derived neurotrophic factor (BDNF). In conclusion, the knock-down of CD24 in astrocytes suppressed hippocampal neuron growth, in which the SHP2-ERK signaling pathway and BNDF were possibly involved.

Effects of ethanol-and choline-treated astrocytes on hippocampal neuron neurite outgrowth in vitro.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

Dexmedetomidine Alleviates LPS-Induced Neuronal Dysfunction by Modulating the AKT/GSK-3β/CRMP-2 Pathway in Hippocampal Neurons.

ObjectiveDexmedetomidine, an α2-adrenergic receptor agonist, mitigates cognitive dysfunction in elderly patients after surgery with general anesthesia. However, the underlying mechanism by which dexmedetomidine reduces cognitive dysfunction remains to be fully elucidated. The aim of this study was to investigate the effects of dexmedetomidine on lipopolysaccharide (LPS)-induced neuronal dysfunction in cultured hippocampal neurons.MethodsLPS, in the presence and absence of dexmedetomidine, was applied to cultured hippocampal neurons to mimic post-surgical inflammation. Neuronal morphology, including neurite outgrowth and synaptic transmission, was observed, and miniature excitatory postsynaptic currents were recorded by electrophysiological patch-clamp.ResultsLPS significantly impaired neurite outgrowth in hippocampal neurons in a concentration- and time-dependent manner, which was reversed by dexmedetomidine treatment. Electrophysiological patch-clamp results showed that LPS induced synaptic transmission dysfunction, which was restored after dexmedetomidine addition. Furthermore, Western blotting assays showed that LPS suppressed the AKT/GSK-3β/CRMP-2 signaling pathway and dexmedetomidine countered the inhibitory effect of LPS by re-activating this pathway.ConclusionIn general, dexmedetomidine protected against the effects of LPS-induced hippocampal neuron damage, including neurite outgrowth and synaptic transmission. Overall, dexmedetomidine modulated the AKT/GSK-3β/CRMP-2 signaling pathway to alleviate LPS-induced neurological dysfunction.

OUP accepted manuscript

The permanent disability after the central nervous system (CNS) injury is due to the weakened regeneration ability of the damaged axon, resulting in the loss of the rebuilding functional relationship with the original targets. One determinant of successful axon regeneration is the activation of intrinsic axon growth ability of injured neurons. MicroRNAs are important epigenetic factors controlling axon regeneration. Here, we demonstrated that the expression of miR-26a in hippocampal neurons is upregulated developmentally. Inhibitions of endogenous miR-26a suppressed the axon growth in hippocampal neurons, and overexpression of miR-26a promoted its axon growth. We also found that the overexpression of miR-26a in retinal ganglion cells also promoted retinal ganglion cells’ survival and optic nerve regeneration. Moreover, endogenous miR-26a promotes the hippocampal neuronal axon growth by suppressing phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression. Thus, our results suggested that the miR-26a‒PTEN pathway regulates CNS axon growth. Collectively, the study not only reveals a new mechanism underlying mammalian axon regeneration but also expands the pool of potential targets that can be manipulated to enhance CNS axon regeneration.

Krüppel-Like Factors 9 and 13 Block Axon Growth by Transcriptional Repression of Key Components of the cAMP Signaling Pathway

Krüppel-like factors (KLFs) are zinc finger transcription factors implicated in diverse biological processes, including differentiation of neural cells. The ability of mammalian neurons to elongate axons decreases during postnatal development in parallel with a decrease in cAMP, and increase in expression of several Klf genes. The paralogous KLFs 9 and 13 inhibit neurite outgrowth, and we hypothesized that their actions are mediated through repression of cAMP signaling. To test this we used the adult mouse hippocampus-derived cell line HT22 engineered to control expression of Klf9 or Klf13 with doxycycline, or made deficient for these Klfs by CRISPR/Cas9 genome editing. We also used primary hippocampal cells isolated from wild type, Klf9–/– and Klf13–/– mice. Forced expression of Klf9 or Klf13 in HT22 changed the mRNA levels of several genes involved with cAMP signaling; the predominant action was gene repression, and KLF13 influenced ∼4 times more genes than KLF9. KLF9 and KLF13 repressed promoter activity of the protein kinase a catalytic subunit alpha gene in transfection-reporter assays; KLF13, but not KLF9 repressed the calmodulin 3 promoter. Forskolin activation of a cAMP-dependent promoter was reduced after forced expression of Klf9 or Klf13, but was enhanced in Klf gene knockout cells. Forced expression of Klf9 or Klf13 blocked cAMP-dependent neurite outgrowth in HT22 cells, and axon growth in primary hippocampal neurons, while Klf gene knockout enhanced the effect of elevated cAMP. Taken together, our findings show that KLF9 and KLF13 inhibit neurite/axon growth in hippocampal neurons, in part, by inhibiting the cAMP signaling pathway.

The lncRNA MALAT1/miR-30/Spastin Axis Regulates Hippocampal Neurite Outgrowth.

Spastin, a microtubule-severing enzyme, is important for neurite outgrowth. However, the mechanisms underlying the post-transcriptional regulation of spastin during microtubule-related processes are largely unknown. We demonstrated that the spastin expression level is controlled by a long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-30 (miR-30) axis during neurite outgrowth. The miR-30 expression level decreased in hippocampal neurons with increasing days in culture, and miR-30 overexpression suppressed while miR-30 inhibition promoted neurite outgrowth in hippocampal neurons. Spastin was validated as a target gene of miR-30 using the luciferase reporter assay. The protein expression, microtubule severing activity, and neurite promoting effect of spastin were suppressed by the overexpression of miR-30 mimics and increased by miR-30 inhibitors. MALAT1 expression increased during neurite outgrowth and MALAT1 silencing impaired neurite outgrowth. miR-30 was a sponge target of MALAT1 and MALAT1/miR-30 altered neurite outgrowth in hippocampal neurons. MALAT1 overexpression reversed the inhibitory effect of miR-30 on the activity of a luciferase reporter construct containing spastin, as well as spastin mRNA and protein expression, indicating that spastin was a downstream effector of MALAT1/miR-30. The MALAT1/miR-30 cascade also modulated spastin-induced microtubule severing, and the MALAT1/miR-30/spastin axis regulated neurite outgrowth in hippocampal neurons. This study suggests a new mechanism governing neurite outgrowth in hippocampal neurons involving MALAT1/miR-30-regulated spastin expression.

The Vps52 subunit of the GARP and EARP complexes is a novel Arf6-interacting protein that negatively regulates neurite outgrowth of hippocampal neurons

ADP ribosylation factor 6 (Arf6) is a small GTP-binding protein implicated in neuronal morphogenesis through endosomal trafficking and actin remodeling. In this study, we identified Vps52, a core subunit of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, as a novel Arf6-binding protein by yeast two-hybrid screening. Vps52 interacted specifically with GTP-bound Arf6 among the Arf family. Immunohistochemical analyses of hippocampal pyramidal cells revealed that fine punctate immunolabeling for Vps52 was distributed throughout neuronal compartments, most densely in the cell body and dendritic shafts, and was largely associated with trans-Golgi network and vesicular endomembranes. In cultured hippocampal neurons, knockdown of Vps52 increased total length of axons and dendrites; these phenotypes were completely restored by co-expression of shRNA-resistant full-length Vps52. However, co-expression of a Vps52 mutant lacking the ability to interact with Arf6 restored only the Vps52-knockdown phenotype of the dendritic length. The present findings suggest that Vps52 is a novel Arf6-interacting protein that regulates neurite outgrowth in hippocampal neurons.

TRPC5 regulates axonal outgrowth in developing retinal ganglion cells

The TRPC5 ion channel is activated upon depletion of intracellular calcium stores, as well as by various stimuli such as nitric oxide (NO), membrane stretch, and cold temperatures. TRPC5 is abundantly expressed in the central nervous system where it has important neuronal functions. In the chick retina, TRPC5 expression was shown to be restricted to amacrine cells (ACs) and Müller glial cells, although its expression was also observed in the ganglion cell layer (GCL) in displaced ACs, as determined by their characteristic cell morphology. However, it is possible that this expression analysis alone might be insufficient to fully understand the expression of TRPC5 in retinal ganglion cells (RGCs). Hence, we analyzed TRPC5 expression by in situ hybridization and immunostaining in the developing mouse retina, and for the first time identified that developing and mature RGCs strongly express TRPC5. The expression begins at E14.5, and is restricted to ACs and RGCs. It was reported that TRPC5 negatively regulates axonal outgrowth in hippocampal neurons. We thus hypothesized that TRPC5 might have similar functions in RGCs since they extend very long axons toward the brain, and this characteristic significantly differs from other retinal cell types. To elucidate its possible involvement in axonal outgrowth, we inhibited TRPC5 activity in developing RGCs which significantly increased RGC axon length. In contrast, overexpression of TRPC5 inhibited axonal outgrowth in developing RGCs. These results indicate that TRPC5 is an important negative regulator of RGC axonal outgrowth. Since TRPC5 is a mechanosensor, it might function to sense abnormal intraocular pressure changes, and could contribute to the death of RGCs in diseases such as glaucoma. In this case, excessive Ca2+ entry through TRPC5 might induce dendritic and axonal remodeling, which could lead to cell death, as our findings clearly indicate that TRPC5 is an important regulator of neurite remodeling.

Insulin-regulated aminopeptidase inhibitor-mediated increases in dendritic spine density are facilitated by glucose uptake.

Ethyl2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate (HFI-419), the benzopyran-based inhibitor of insulin-regulated aminopeptidase (IRAP), has previously been shown to improve spatial working and recognition memory in rodents. However, the mechanism of its cognitive-enhancing effect remains unknown. There is a close correlation between dendritic spine density and learning in vivo and several studies suggest that increases in neuronal glucose uptake and/or alterations to the activity of matrix metalloproteinases (MMPs) may improve memory and increase dendritic spine density. We aimed to identify the potential mechanism by which HFI-419 enhances memory by utilizing rat primary cultures of hippocampal cells. Alterations to dendritic spine density were assessed in the presence of varying concentrations of HFI-419 at different stages of hippocampal cell development. In addition, glucose uptake and changes to spine density were assessed in the presence of indinavir, an inhibitor of the glucose transporter 4 (GLUT4 ), or the matrix metalloprotease inhibitor CAS 204140-01-2. We confirmed that inhibition of IRAP activity with HFI-419 enhanced spatial working memory in rats, and determined that this enhancement may be driven by GLUT4 -mediated changes to dendritic spine density. We observed that IRAP inhibition increased dendritic spine density prior to peak dendritic growth in hippocampal neurons, and that spine formation was inhibited when GLUT4 -mediated glucose uptake was blocked. In addition, during the peak phase of dendritic spine growth, the effect of IRAP inhibition on enhancement of dendritic spine density resulted specifically in an increase in the proportion of mushroom/stubby-like spines, a morphology associated with memory and learning. Moreover, these spines were deemed to be functional based on their expression of the pre-synaptic markers vesicular glutamate transporter 1 and synapsin. Overall, or findings suggest that IRAP inhibitors may facilitate memory by increasing hippocampal dendritic spine density via a GLUT4 -mediated mechanism. Cover Image for this issue: doi: 10.1111/jnc.14745.

Volatile Oil from Acorus gramineus Ameliorates the Injury Neurons in the Hippocampus of Amyloid Beta 1-42 Injected Mice.

In recent years, the extraction fraction of volatile oil from Acorus gramineus has significant effects on anti-dementia and improving the learning and memory of animals. To date, limited studies have determined whether volatile oil from A. gramineus has the protective effect on neuronal damage. The aim of this study was to investigate the protective effects of volatile oil from A. gramineus on Alzheimer's disease (AD) mice, by means of behavior test, immunohistochemistry and western blot methods. In this study, mice were injected with Aβ1-42 in the bilateral hippocampus to establish the AD model. On the seventh day after modeling, the mice with cognitive dysfunction were selected by the novel object recognition task. Subsequently, the volatile oil treatment groups underwent intragastric administration for per 10 g body weight 2.5 or 5 μL volatile oil from A. gramineus for 3 weeks. The control group and the AD group were given the same amount of saline. Our results showed that after treatment of volatile oil from A. gramineus, the number of Doublecortin and Nestin positive cells increased significantly, suggesting that the volatile oil from A. gramineus may induce the regeneration of hippocampal neurons in mice, and promote the growth of hippocampal neurons by upregulation of brain-derived neurotrophic factor, tyrosine protein kinase B, and neurotrophin-3 expression. These results might provide more experimental evidences for underlying mechanism about the neuroprotective effects of volatile oil from A. gramineus against AD relevant symptoms. Anat Rec, 302:2261-2270, 2019. © 2019 American Association for Anatomy.

Role of serotonin 4 receptor in the growth of hippocampal neurons during the embryonic development in mice

Serotonin (5-HT) homeostasis is critical for the brain development which influences neurogenesis, neuronal migration, and circuit formation. Distinctive distribution patterns of serotonin receptors (5-HTRs) in the brain govern various physiological activities. Amongst the 5-HTRs, serotonin 4 receptor (5-HT4R) is widely expressed in embryonic forebrain and affects neuronal development, synaptogenesis, and behavior, but its specific role in brain development is still not completely understood. Therefore, in the present study, we addressed the roles of 5-HT4R in the growth of hippocampal neurons during the development of mice brain. We cultured hippocampal neurons of the mouse at embryonic day 18 and then treatment of 5-HT4R agonist RS67333 was employed. We found RS67333 significantly increased the axonal length, diameter and branching along with total dendritic length, number of primary dendrites and their branching. In addition, these effects were neutralized by the concomitant treatment of 5-HT4R antagonist GR125487, which confirmed the specific role of the 5-HT4R in the growth of axon and dendrites. Further, the treatment of RS67333 upregulated the mRNA expression of collapsin response mediator protein-2 (CRMP2) and non-phosphorylated CRMP2 (npCRMP2) together with neurotrophic factors (BDNF, NT-3, NGF) and TRK-A. Additionally, the current research findings reveal that the knockdown of CRMP2 inhibited RS67333-induced growth of the axons and dendrites, which indicates that CRMP2 is required for the 5-HT4R-mediated growth of the axons and dendrites. Overall, the findings of the present in vitro study enrich the understanding and provide insight roles of 5-HT4R in embryonic brain development by promoting the growth of hippocampal neurons.