Adult expression of the cell adhesion protein Fasciclin 3 is required for the maintenance of adult olfactory interneurons.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3-mediated cell death in olfactory local interneurons, which can be rescued by overexpressing baculovirus p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a crucial role for axon guidance proteins in the maintenance of neuronal circuits in adults.

Prolonged myelin deficits contribute to neuron loss and functional impairments after ischaemic stroke.

Ischaemic stroke causes neuron loss and long-term functional deficits. Unfortunately, effective approaches to preserving neurons and promoting functional recovery remain unavailable. Oligodendrocytes, the myelinating cells in the CNS, are susceptible to oxygen and nutrition deprivation and undergo degeneration after ischaemic stroke. Technically, new oligodendrocytes and myelin can be generated by the differentiation of oligodendrocyte precursor cells (OPCs). However, myelin dynamics and their functional significance after ischaemic stroke remain poorly understood. Here, we report numerous denuded axons accompanied by decreased neuron density in sections from ischaemic stroke lesions in human brain, suggesting that neuron loss correlates with myelin deficits in these lesions. To investigate the longitudinal changes in myelin dynamics after stroke, we labelled and traced pre-existing and newly-formed myelin, respectively, using cell-specific genetic approaches. Our results indicated massive oligodendrocyte death and myelin loss 2 weeks after stroke in the transient middle cerebral artery occlusion (tMCAO) mouse model. In contrast, myelin regeneration remained insufficient 4 and 8 weeks post-stroke. Notably, neuronal loss and functional impairments worsened in aged brains, and new myelin generation was diminished. To analyse the causal relationship between remyelination and neuron survival, we manipulated myelinogenesis by conditional deletion of Olig2 (a positive regulator) or muscarinic receptor 1 (M1R, a negative regulator) in OPCs. Deleting Olig2 inhibited remyelination, reducing neuron survival and functional recovery after tMCAO. Conversely, enhancing remyelination by M1R conditional knockout or treatment with the pro-myelination drug clemastine after tMCAO preserved white matter integrity and neuronal survival, accelerating functional recovery. Together, our findings demonstrate that enhancing myelinogenesis is a promising strategy to preserve neurons and promote functional recovery after ischaemic stroke.

Transplantation of dorsal root ganglia overexpressing the NaChBac sodium channel improves locomotion after complete SCI.

Spinal cord injury (SCI) is a debilitating condition currently lacking treatment. Severe SCI causes the loss of most supraspinal inputs and neuronal activity caudal to the injury, which, coupled with the limited endogenous capacity for spontaneous regeneration, can lead to complete functional loss even in anatomically incomplete lesions. We hypothesized that transplantation of mature dorsal root ganglia (DRGs) genetically modified to express the NaChBac sodium channel could serve as a therapeutic option for functionally complete SCI. We found that NaChBac expression increased the intrinsic excitability of DRG neurons and promoted cell survival and neurotrophic factor secretion in vitro. Transplantation of NaChBac-expressing dissociated DRGs improved voluntary locomotion 7 weeks after injury compared to control groups. Animals transplanted with NaChBac-expressing DRGs also possessed higher tubulin-positive neuronal fiber and myelin preservation, although serotonergic descending fibers remained unaffected. We observed early preservation of the corticospinal tract 14 days after injury and transplantation, which was lost 7 weeks after injury. Nevertheless, transplantation of NaChBac-expressing DRGs increased the neuronal excitatory input by an increased number of VGLUT2 contacts immediately caudal to the injury. Our work suggests that the transplantation of NaChBac-expressing dissociated DRGs can rescue significant motor function, retaining an excitatory neuronal relay activity immediately caudal to injury.

Impacts of methotrexate on survival, dendrite development, and synapse formation of cortical neurons.

Methotrexate (MTX) is an anti-metabolite that has been used for the treatment of patients of acute lymphocytic leukemia or non-Hodgikin lymphoma for decades. In some cases, MTX-treated patients suffer from neurological side effects, including seizures and cognitive dysfunctions. While most patients are at developmental stages, information of the mechanisms of the side effects of MTX treatment on the developing neurons has been limited. Neurons develop in five steps in the human brain: neurogenesis, polarity formation, dendrite and axon development, synapse formation, and neuronal death. Except for neurogenesis, these processes can be recapitulated in the primary culture system of cortical neurons. Using primary cultured cortical neurons, we studied the impact of MTX treatment on dendrite development, synapse formation, and neuronal death in the present report. MTX treatment impaired neuronal survival, dendrite development, and synapse formation. Interestingly, half maximal effective concentrations (EC50 s) of MTX for all three processes are at the similar range and lower than the MTX concentration in the cerebrospinal fluid in treated patients. Our results provide possible mechanisms of neurological side effects in treated patients.

New insights into sodium phenylbutyrate as a pharmacotherapeutic option for neurological disorders.

Neurological disorders (NDs) are diseases of the central and peripheral nervous systems that affect more than one billion people worldwide. The risk of developing an ND increases with age due to the vulnerability of the different organs and systems to genetic, environmental, and social changes that consequently cause motor and cognitive deficits that disable the person from their daily activities and individual and social productivity. Intrinsic factors (genetic factors, age, gender) and extrinsic factors (addictions, infections, or lifestyle) favor the persistence of systemic inflammatory processes that contribute to the evolution of NDs. Neuroinflammation is recognized as a common etiopathogenic factor of ND. The study of new pharmacological options for the treatment of ND should focus on improving the characteristic symptoms and attacking specific molecular targets that allow the delay of damage processes such as neuroinflammation, oxidative stress, cellular metabolic dysfunction, and deregulation of transcriptional processes. In this review, we describe the possible role of sodium phenylbutyrate (NaPB) in the pathogenesis of Alzheimer's disease, hepatic encephalopathy, aging, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis; in addition, we describe the mechanism of action of NaPB and its beneficial effects that have been shown in various in vivo and in vitro studies to delay the evolution of any ND.

Enhancement of neurogenesis and cognition through intranasal co-delivery of galanin receptor 2 (GALR2) and neuropeptide Y receptor 1 (NPY1R) agonists: a potential pharmacological strategy for cognitive dysfunctions.

BACKGROUND: Spatial memory deficits and reduced neuronal survival contribute to cognitive decline seen in the aging process. Current treatments are limited, emphasizing the need for innovative therapeutic strategies. This research explored the combined effects of intranasally co-administered galanin receptor 2 (GALR2) and neuropeptide Y1 receptor (NPY1R) agonists, recognized for their neural benefits, on spatial memory, neuronal survival, and differentiation in adult rats. After intranasal co-delivery of the GALR2 agonist M1145 and a NPY1R agonist to adult rats, spatial memory was tested with the object-in-place task 3 weeks later. We examined neuronal survival and differentiation by assessing BrdU-IR profiles and doublecortin (DCX) labeled cells, respectively. We also used the GALR2 antagonist M871 to confirm GALR2's crucial role in promoting cell growth. RESULTS: Co-administration improved spatial memory and increased the survival rate of mature neurons. The positive effect of GALR2 in cell proliferation was confirmed by the nullifying effects of its antagonist. The treatment boosted DCX-labeled newborn neurons and altered dendritic morphology, increasing cells with mature dendrites. CONCLUSIONS: Our results show that intranasal co-delivery of GALR2 and NPY1R agonists improves spatial memory, boosts neuronal survival, and influences neuronal differentiation in adult rats. The significant role of GALR2 is emphasized, suggesting new potential therapeutic strategies for cognitive decline.

A novel anti-apoptotic role for Cdc42/ACK-1 signaling in neurons.

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.

Inhibition of Apoptosis in a Model of Ischemic Stroke Leads to Enhanced Cell Survival, Endogenous Neural Precursor Cell Activation and Improved Functional Outcomes.

Stroke results in neuronal cell death, which causes long-term disabilities in adults. Treatment options are limited and rely on a narrow window of opportunity. Apoptosis inhibitors demonstrate efficacy in improving neuronal cell survival in animal models of stroke. However, many inhibitors non-specifically target apoptosis pathways and high doses are needed for treatment. We explored the use of a novel caspase-3/7 inhibitor, New World Laboratories (NWL) 283, with a lower IC50 than current caspase-3/7 inhibitors. We performed in vitro and in vivo assays to determine the efficacy of NWL283 in modulating cell death in a preclinical model of stroke. In vitro and in vivo assays show that NWL283 enhances cell survival of neural precursor cells. Delivery of NWL283 following stroke enhances endogenous NPC migration and leads to increased neurogenesis in the stroke-injured cortex. Furthermore, acute NWL283 administration is neuroprotective at the stroke injury site, decreasing neuronal cell death and reducing microglia activation. Coincident with NWL283 delivery for 8 days, stroke-injured mice exhibited improved functional outcomes that persisted following cessation of the drug. Therefore, we propose that NWL283 is a promising therapeutic warranting further investigation to enhance stroke recovery.

Development of Pleiotropic TrkB and 5-HT4 Receptor Ligands as Neuroprotective Agents.

One common event that is the most detrimental in neurodegenerative disorders, even though they have a complex pathogenesis, is the increased rate of neuronal death. Endogenous neurotrophins consist of the major neuroprotective factors, while brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor TrkB are described in a number of studies for their important neuronal effects. Normal function of this receptor is crucial for neuronal survival, differentiation, and synaptic function. However, studies have shown that besides direct activation, the TrkB receptor can be transactivated via GPCRs. It has been proven that activation of the 5-HT4 receptor and transactivation of the TrkB receptor have a positive influence on neuronal differentiation (total dendritic length, number of primary dendrites, and branching index). Because of that and based on the main structural characteristics of LM22A-4, a known activator of the TrkB receptor, and RS67333, a partial 5-HT4 receptor agonist, we have designed and synthesized a small data set of novel compounds with potential dual activities in order to not only prevent neuronal death, but also to induce neuronal differentiation in neurodegenerative disorders.

Early-onset brain alterations during postnatal development in a mouse model of CDKL5 deficiency disorder.

Mutations in the CDKL5 gene are the cause of CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental condition characterized by early-onset epilepsy, motor impairment, intellectual disability, and autistic features. A mouse model of CDD, the Cdkl5 KO mouse, that recapitulates several aspects of CDD symptomology, has helped to highlight brain alterations leading to CDD neurological defects. Studies of brain morphogenesis in adult Cdkl5 KO mice showed defects in dendritic arborization of pyramidal neurons and in synaptic connectivity, a hypocellularity of the hippocampal dentate gyrus, and a generalized microglia over-activation. Nevertheless, no studies are available regarding the presence of these brain alterations in Cdkl5 KO pups, and their severity in early stages of life compared to adulthood. A deeper understanding of the CDKL5 deficient brain during an early phase of postnatal development would represent an important milestone for further validation of the CDD mouse model, and for the identification of the optimum time window for treatments that target defects in brain development. In sight of this, we comparatively evaluated the dendritic arborization and spines of cortical pyramidal neurons, cortical excitatory and inhibitory connectivity, microglia activation, and proliferation and survival of granule cells of the hippocampal dentate gyrus in hemizygous Cdkl5 KO male (-/Y) mice aged 7, 14, 21, and 60 days. We found that most of the structural alterations in Cdkl5 -/Y brains are already present in pups aged 7 days and do not worsen with age. In contrast, the difference in the density of excitatory and inhibitory terminals between Cdkl5 -/Y and wild-type mice changes with age, suggesting an age-dependent cortical excitatory/inhibitory synaptic imbalance. Confirming the precocious presence of brain defects, Cdkl5 -/Y pups are characterized by an impairment in neonatal sensory-motor reflexes.

NOD-like receptor NLRC5 promotes neuroinflammation and inhibits neuronal survival in Parkinson's disease models.

Parkinson's disease (PD) is mainly characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and neuroinflammation mediated by overactivated microglia and astrocytes. NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5) has been reported to participate in various immune disorders, but its role in neurodegenerative diseases remains unclear. In the current study, we found that the expression of NLRC5 was increased in the nigrostriatal axis of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced PD, as well as in primary astrocytes, microglia and neurons exposed to different neurotoxic stimuli. In an acute MPTP-induced PD model, NLRC5 deficiency significantly reduced dopaminergic system degeneration and ameliorated motor deficits and striatal inflammation. Furthermore, we found that NLRC5 deficiency decreased the expression of the proinflammatory genes IL-1ß, IL-6, TNF-α and COX2 in primary microglia and primary astrocytes treated with neuroinflammatory stimuli and reduced the inflammatory response in mixed glial cells in response to LPS treatment. Moreover, NLRC5 deficiency suppressed activation of the NF-κB and MAPK signaling pathways and enhanced the activation of AKT-GSK-3ß and AMPK signaling in mixed glial cells. Furthermore, NLRC5 deficiency increased the survival of primary neurons treated with MPP+ or conditioned medium from LPS-stimulated mixed glial cells and promoted activation of the NF-κB and AKT signaling pathways. Moreover, the mRNA expression of NLRC5 was decreased in the blood of PD patients compared to healthy subjects. Therefore, we suggest that NLRC5 promotes neuroinflammation and dopaminergic degeneration in PD and may serve as a marker of glial activation.

The role of protein glycosylation in the occurrence and outcome of acute ischemic stroke.

Acute ischemic stroke (AIS) is a serious and life-threatening disease worldwide. Despite thrombolysis or endovascular thrombectomy, a sizeable fraction of patients with AIS have adverse clinical outcomes. In addition, existing secondary prevention strategies with antiplatelet and anticoagulant drugs therapy are not able to adequately decrease the risk of ischemic stroke recurrence. Thus, exploring novel mechanisms for doing so represents an urgent need for the prevention and treatment of AIS. Recent studies have discovered that protein glycosylation plays a critical role in the occurrence and outcome of AIS. As a common co- and post-translational modification, protein glycosylation participates in a wide variety of physiological and pathological processes by regulating the activity and function of proteins or enzymes. Protein glycosylation is involved in two causes of cerebral emboli in ischemic stroke: atherosclerosis and atrial fibrillation. Following ischemic stroke, the level of brain protein glycosylation becomes dynamically regulated, which significantly affects stroke outcome through influencing inflammatory response, excitotoxicity, neuronal apoptosis, and blood-brain barrier disruption. Drugs targeting glycosylation in the occurrence and progression of stroke may represent a novel therapeutic idea. In this review, we focus on possible perspectives about how glycosylation affects the occurrence and outcome of AIS. We then propose the potential of glycosylation as a therapeutic drug target and prognostic marker for AIS patients in the future.

The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions.

Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1's KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions.

Activation of the PI3K/AKT/mTOR Pathway in Cajal-Retzius Cells Leads to Their Survival and Increases Susceptibility to Kainate-Induced Seizures.

Cajal-Retzius cells (CRs) are a class of transient neurons in the mammalian cortex that play a critical role in cortical development. Neocortical CRs undergo almost complete elimination in the first two postnatal weeks in rodents and the persistence of CRs during postnatal life has been detected in pathological conditions related to epilepsy. However, it is unclear whether their persistence is a cause or consequence of these diseases. To decipher the molecular mechanisms involved in CR death, we investigated the contribution of the PI3K/AKT/mTOR pathway as it plays a critical role in cell survival. We first showed that this pathway is less active in CRs after birth before massive cell death. We also explored the spatio-temporal activation of both AKT and mTOR pathways and reveal area-specific differences along both the rostro-caudal and medio-lateral axes. Next, using genetic approaches to maintain an active pathway in CRs, we found that the removal of either PTEN or TSC1, two negative regulators of the pathway, lead to differential CR survivals, with a stronger effect in the Pten model. Persistent cells in this latter mutant are still active. They express more Reelin and their persistence is associated with an increase in the duration of kainate-induced seizures in females. Altogether, we show that the decrease in PI3K/AKT/mTOR activity in CRs primes these cells to death by possibly repressing a survival pathway, with the mTORC1 branch contributing less to the phenotype.

The Role of MEF2 Transcription Factor Family in Neuronal Survival and Degeneration.

The family of myocyte enhancer factor 2 (MEF2) transcription factors comprises four highly conserved members that play an important role in the nervous system. They appear in precisely defined time frames in the developing brain to turn on and turn off genes affecting growth, pruning and survival of neurons. MEF2s are known to dictate neuronal development, synaptic plasticity and restrict the number of synapses in the hippocampus, thus affecting learning and memory formation. In primary neurons, negative regulation of MEF2 activity by external stimuli or stress conditions is known to induce apoptosis, albeit the pro or antiapoptotic action of MEF2 depends on the neuronal maturation stage. By contrast, enhancement of MEF2 transcriptional activity protects neurons from apoptotic death both in vitro and in preclinical models of neurodegenerative diseases. A growing body of evidence places this transcription factor in the center of many neuropathologies associated with age-dependent neuronal dysfunctions or gradual but irreversible neuron loss. In this work, we discuss how the altered function of MEF2s during development and in adulthood affecting neuronal survival may be linked to neuropsychiatric disorders.

Interaction of L1CAM with LC3 Is Required for L1-Dependent Neurite Outgrowth and Neuronal Survival.

The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.

Ligand-Induced Activation of GPR110 (ADGRF1) to Improve Visual Function Impaired by Optic Nerve Injury.

It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.

The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions.

The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions.

GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.

Survival and functional recovery of primary cortical neurons exposed to actinomycin D.

Actinomycin D (ActD) is an antineoplastic antibiotic that has been commonly used for the treatment of various tumors, including Wilms' tumor, rhabdomyosarcoma, and gestational trophoblastic neoplasia. Recent studies have proposed actinomycin D (ActD) as a novel therapeutic candidate for glioblastoma. ActD significantly reduces tumor growth in recurrent glioblastoma patient-derived mouse models and increases survival by downregulating SOX2 expression. However, ActD treatment of brain tumors can lead to unnecessary exposure of surrounding neurons and normal glial cells to ActD. Cellular and molecular studies are required to estimate and minimize the neurological side effects of ActD. This study investigated the short- and long-term toxicological responses of the primary cortical neurons to ActD. We examined concentration-dependent survival of primary cortical neurons and differential susceptibilities of excitatory, inhibitory neurons, and glial cells to ActD. Distinct alterations in intracellular signaling pathways in cortical neurons were also studied when exposed to ActD. Importantly, we found that primary cortical neurons after ActD discontinuation showed active intracellular signaling pathways responding to extracellular neurotropic factors, but they had extremely poor transcription activity reversibility that was inhibited even by 30-min low-dose ActD exposure. These findings indicate the direct toxicity and extremely poor reversibility of ActD in neurons during chemotherapy for brain tumors.