Exportin 4 depletion leads to nuclear accumulation of a subset of circular RNAs.

Numerous RNAs are exported from the nucleus, abnormalities of which lead to cellular complications and diseases. How thousands of circular RNAs (circRNAs) are exported from the nucleus remains elusive. Here, we provide lines of evidence to demonstrate a link between the conserved Exportin 4 (XPO4) and nuclear export of a subset of circRNAs in metazoans. Exonic circRNAs (ecircRNAs) with higher expression levels, larger length, and lower GC content are more sensitive to XPO4 deficiency. Cellular insufficiency of XPO4 leads to nuclear circRNA accumulation, circRNA:DNA (ciR-loop) formation, linear RNA:DNA (liR-loop) buildup, and DNA damage. DDX39 known to modulate circRNA export can resolve ciR-loop, and splicing factors involved in the biogenesis of circRNAs can also affect the levels of ciR-loop. Testis and brain are two organs with high abundance of circRNAs, and insufficient XPO4 levels are detrimental, as Xpo4 heterozygous mice display male infertility and neural phenotypes. Increased levels of ciR-loop, R-loop, and DNA damage along with decreased cell numbers are observed in testis and hippocampus of Xpo4 heterozygotes. This study sheds light on the understandings of mechanism of circRNA export and reveals the significance of efficient nuclear export of circRNAs in cellular physiology.

Alterations of serine racemase expression determine proliferation and differentiation of neuroblastoma cells.

Although the role of serine racemase (SR) in neuropsychiatric disorders has been extensively studied, its role in cell proliferation and differentiation remains unclear. Deletion of Srr, the encoding gene for SR, has been shown to reduce dendritic arborization and dendritic spine density in the brains of adult mice, whereas increased SR levels have been associated with differentiation in cell cultures. Previously, we demonstrated that valproic acid induces differentiation in the N2A neuroblastoma cell line, and that this differentiation is associated with increased SR expression. These observations suggest that SR may have a role in cell proliferation and differentiation. We herein found that both valproic acid and all-trans retinoic acid induced N2A differentiation. In contrast, knockdown of SR reduced levels of differentiation, increased N2A proliferation, promoted cell cycle entry, and modulated expression of cell cycle-related proteins. To further evaluate the effects of SR expression on cell proliferation and differentiation, we used an in vivo model of neuroblastoma in nude mice. N2A cells stably expressing scramble shRNA (Srrwt -N2A) or specific Srr shRNA (Srrkd -N2A) were subcutaneously injected into nude mice. The weights and volumes of Srrwt -N2A-derived tumors were lower than Srrkd -N2A-derived tumors. Furthermore, Srrwt -N2A-derived tumors were significantly mitigated by intraperitoneal injection of valproic acid, whereas Srrkd -N2A-derived tumors were unaffected. Taken together, our findings demonstrate for the first time that alterations in SR expression determine the transition between proliferation and differentiation in neural progenitor cells. Thus, in addition to its well-established roles in neuropsychiatric disorders, our study has highlighted a novel role for SR in cell proliferation and differentiation.

Serine Racemase Expression Differentiates Aging from Alzheimer's Brain.

Aging is an inevitable process characterized by progressive loss of physiological integrity and increased susceptibility to cancer, diabetes, cardiovascular, and neurodegenerative diseases; aging is the primary risk factor for Alzheimer's disease (AD), the most common cause of dementia. AD is characterized by brain pathology, including extracellular deposition of amyloid aggregation and intracellular accumulation of neurofibrillary tangles composed of hyperphosphorylated tau protein. In addition, losses of synapses and a wide range of neurons are pivotal pathologies in the AD brain. Accumulating evidence demonstrates hypoactivation of hippocampal neural networks in the aging brain, whereas AD-related mild cognitive impairment (AD-MCI) begins with hyperactivation, followed by a diminution of hippocampal activity as AD develops. The biphasic trends of the activity of the hippocampal neural network are consistent with the alteration of N-methyl-D-aspartate receptor (NMDA-R) activity from aging to prodromal (AD-MCI) to mid-/late stage AD. D-serine, a product of racemization catalyzed by serine racemase (SR), is an important co-agonist of the NMDA-R which is involved in synaptic events including neurotransmission, synaptogenesis, long-term potentiation (LTP), development, and excitotoxicity. SR and D-serine are decreased in the hippocampus of the aging brain, correlating with impairment of cognitive function. By contrast, SR is increased in AD brain, which is associated with a greater degree of cognitive dysfunction. Emerging studies suggest that D-serine levels in the brain or in cerebral spinal fluid from AD patients are higher than in age-matched controls, but the results are inconsistent. Very recently, serum D-serine levels in AD were reported to correlate with sex and clinical dementia rating (CDR) stage. This review will discuss alterations of NMDA-R and SR in aging and AD brain, and the mechanisms underlying the differential regulation of SR will be probed. Collectively, we propose that SR may be a molecular switch that distinguishes the effects of aging from those of AD on the brain.

Deletion of serine racemase reverses neuronal insulin signaling inhibition by amyloid-ß oligomers.

Dysregulation of insulin signaling in the Alzheimer's disease (AD) brain has been extensively reported. Serine racemase (SR) modulates insulin secretion in pancreatic islets. This study aimed to examine whether SR regulates insulin synthesis and secretion in neurons, thereby modulating insulin signaling in the AD brain. Srr-knockout (Srr-/- ) mice generated with the CRISPR/Cas9 technique were used. Using immunofluorescence and fluorescence in situ hybridization, levels of insulin protein and insulin(ins2) mRNA were significantly increased in the hippocampal but not in hypothalamic sections of Srr-/- mice compared with WT mice. Real-time quantitative PCR revealed that ins2 mRNA from primary hippocampal neuronal cultures of Srr-/- mice was significantly increased compared with that from cultured neurons of WT mice. Notably, the secretion of proinsulin C-peptide was increased in Srr-/- neurons relative to WT neurons. By examining membrane fractional proteins with immunoblotting, Srr-/- neurons retained ATP-dependent potassium channels on plasmalemma and correspondingly contained higher levels of p-AMPK. After treatment with Aß42, the phosphorylation levels of insulin receptor substrate at serine 616 636 (p-IRS1ser616,636 ) were significantly lower, whereas p-AKT308 and p-AKT473 were higher in Srr-/- neurons than in WT neurons, respectively. The phosphorylated form of c-Jun N-terminal kinase decreased in the cultured Srr-/- neurons relative to the WT neurons upon Aß42 treatment. In contrast, phosphorylated protein kinase R remained at the same levels. Further, reactive oxygen species were reduced in cultured Srr-/- neurons under Aß42 treatment relative to the WT neurons. Collectively, our study indicated that Srr deletion promoted insulin synthesis and secretion of proinsulin C-peptide, thereby reversing insulin resistance by Aß42. This study suggests that targeting the neuronal SR may be utilized to enhance insulin signaling which is inhibited at the early stage of the AD brain.

Amyloid peptide exerts a rapid induction of Dicer1 protein in neuron via reducing phosphorylation.

A growing number of evidence suggests that altered microRNA network in the brain contributes to the risk of Alzheimer's disease(AD). Dicer1 is a type III riboendonuclease which cleaves pre-microRNA into functional microRNA. Reduction of Dicer1 or Dicer1 mutation has been involved in cancer, aging or age-related macular degeneration. Recently, we found a possible link between Dicer1 and AD. In particular, Dicer1 protein and Dicer1 mRNA is reduced in the hippocampus and the cortex of an animal model of AD and exposure to Aß42 oligomer(AßO) longer than 6 h reduces the transcription of Dicer1 gene in neuron, via depletion of NF-E2-related factor-2. In this study, exposure to AßO at shorter time increased Dicer1 protein in neuron in a dose-dependent mode; but the mRNA level remained unaltered. Under this treatment regime,AßO reduced phosphorylation level of Dicer1 and of its binding partner, transactivation response element RNA-binding protein(TRBP). Addition of a JNK inhibitor,SP600125, or an ERK inhibitor,U0126, further increased Dicer1 protein compared to Aßo treatment alone, with simultaneaous reduction of phospho-Dicer1, but with different effects on phospho-TRBP. Finally, an inhibitor of calcineurin,FK506, further increased Dicer1 protein compared to Aßo treatment alone. Thus, phosphorylation of Dicer1 and TRBP was determined by mitogen activated protein kinases JNK,ERK, and protein phosphatase 2B(calcineurin) which together determined Dicer1 stability. In summary, reduced phosphorylation of Dicer1 accounted for the rapid induction of Dicer1 by AßO. This study highlights a novel way by which AßO regulates Dicer1.

Repurposing bortezomib for choroidal neovascularization treatment via antagonizing VEGF-A and PDGF-D mediated signaling.

Neovascular age-related macular degeneration (neoAMD) is the leading cause of blindness in AMD and manifests as choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) therapies are the mainstay treatments but with limited efficacy and cause detrimental effects on the retina after long-term application. These disadvantages warrant alternative strategy. Herein, we examined the effect on CNV by intravitreal injection of bortezomib, a reversible proteasome inhibitor, and further dissected the mechanism. Krypton red Laser was used to create CNV model in mice. The angiogenesis volume was assessed in choroidal flat-mount with isolectin GS-IB4 labeling and the leakage was examined with fluorescein fundus angiography. Injection of Borsub inhibited angiogenesis in the CNV model which was dose-dependent; the injection significantly inhibited leakage as well. Furthermore, Borsub injection reduced the contents of VEGF-A, macrophage chemotactic factor 1 (MCP-1), and platelet-derived growth factor (PDGF)-D but not PDGF-B, examined by enzyme-linked immunosorbent assay, in choroid/retinal pigment epithelium (RPE) tissue. These injections also reduced phospho-VEGFR-2 and phospho-PDGFRß in choroid/RPE tissue examined by immunoblotting. Moreover, Borsub inhibited the recruitment of mural cells or macrophages to laser-injured spots. Injection of Borsub indicated negative effect on scotopic and photopic responses recorded by electroretinogram. Altogether, intravitreal injection of Borsub significantly reduced CNV by antagonizing VEGF-A/Flk-1 and PDGF-D/PDGFRß pathways without impacting electroretinography parameters. Thus, Borsub may offer an invaluable therapy for the prevention and treatment of neoAMD.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

Dicer1 promotes Aß clearance via blocking B2 RNA-mediated repression of apolipoprotein E.

Metabolism of ß-amyloid is critical for healthy brain. Decreased clearance of ß-amyloid is associated with ensued accumulation of amyloid peptide, culminating in formation of senile plaques, a neuropathological hallmark of Alzheimer's disease(AD). Apolipoprotein E (APOE), a lipoprotein for phospholipid and cholesterol metabolism, is predominantly synthesized by glia in the central nervous system, controlling Aß aggregation and metabolism. By use of stereotactic injection and a Morris water maze, we found that delivery of Dicer1-expressing adenovirus into the hippocampus of an animal model of AD mice APPswe/PSEN1deltaE9 significantly improved spatial memory. The effect was associated with reduced amyloid peptides in the hippocampus which were analyzed with immunofluorescence and enzyme-linked immunosorbent assay. With western blot, quantitative real-time PCR, fluorescence in situ hybridization, and northern blot,Dicer1 overexpression increased apolipoprotein E (APOE) and concomitantly decreased B2 RNA in the hippocampus of the AD mice and in astrocyte cultures whereas transfection of B2 Mm2 RNA decreased APOE mRNA and protein levels in astrocyte cultures. Further, human or mouse APOE mRNA was found containing Alu RNA or its equivalent, B2 Mm2 RNA, locating downstream of its 3'-untranslated region (UTR), respectively. The 3'-UTR or 3'-UTR in conjunction with the downstream Alu/B2 RNA were cloned into a luciferase reporter; with dual-luciferase assay, we found that simultaneous transfection of Dicer1 siRNA or Alu/B2 RNA decreased the corresponding luciferase activities which suggest that Alu RNA mediated APOE mRNA degradation. Altogether, Dicer1 expression mediated amyloid peptide clearance by increasing APOE via blocking B2 RNA-mediated APOE mRNA degradation.

Brain Dicer1 Is Down-Regulated in a Mouse Model of Alzheimer's Disease Via Aß42-Induced Repression of Nuclear Factor Erythroid 2-Related Factor 2.

Dicer1 is a microRNA-processing enzyme which plays critical roles in neuronal survival and neuritogenesis. Dicer1 deletion induces neurodegeneration or degeneration in retinal pigment epithelium, which is associated with oxidative stress. Oxidative stress is thought to be central in the pathogenesis of Alzheimer's disease (AD). Therefore, we hypothesize that Dicer1 may play roles in AD. Using immunoblotting and quantitative real-time PCR, Dicer1 protein and mRNA were reduced in the hippocampi of the AD mouse model APPswe/PSEN1dE9 compared with littermate controls. SiRNA-mediated Dicer1 knockdown induced oxidative stress and apoptosis and reduced mitochondrial membrane potential in cultured neurons. Chronic Aß42 exposure decreased Dicer1 and nuclear factor erythroid 2-related factor 2 (Nrf2) which were reversed by N-acetyl-cystein. Nrf2 overexpression increased Dicer1 mRNA and protein and reverted the Aß42-induced Dicer1 reduction. We further cloned Dicer1 promoter variants harboring the Nrf2-binding site, the antioxidant response elements (ARE), into a luciferase reporter and found that simultaneous transfection of Nrf2-expressing plasmid increased luciferase expression from these promoter constructs. ChIP assays indicated that Nrf2 directly interacted with the ARE motifs in the Dicer1 promoter. Furthermore, Dicer1 overexpression in cultured neurons reverted Aß42-induced neurite deficits. Notably, injection of Dicer1-expressing adenovirus into the hippocampus of the mice significantly improved spatial learning. Altogether, we found novel roles of Dicer1 in AD and a novel regulatory pathway for Dicer1. These results suggest that Dicer1 is a target in AD therapy, especially at the early stage of this disorder. In this study, we found that Dicer1 was reduced in the brain of AD mice which is the first report to examine Dicer1 in AD. We further found (i) that Aß42 exposure decreased Dicer1 via attenuating Nrf2-ARE signaling and (ii) injection of Dicer1-expressing adenovirus into the hippocampus of the AD mice significantly improved spatial learning. Altogether, we found novel roles of Dicer1 in AD and a novel regulatory pathway for Dicer1. This study may open new avenues for investigating potential pathognomonics and pathogenesis in AD.

Sp4 controls constitutive expression of neuronal serine racemase and NF-E2-related factor-2 mediates its induction by valproic acid.

Serine racemase (SR) synthesizes l-type serine to its enantimor, d-serine which participates in physiological processes and in pathological conditions. In the central nervous system, SR is highly expressed in neurons and astrocytes but expressed at relatively lower amount in microglia. However, the mechanism by which SR is highly expessed in neurons is hitherto unknown. We report that the SR mRNA and protein levels in Neuro-2a were increased by valproic acid (VPA), a neuron differentiation stimulator as well as a histone deacetylase inhibitor. SR proximal promoter contained nine putative Sp-binding elements and in the exon 1, three putative anti-oxidant elements (AREs) were conservative among human, rat, and mouse genome. The promoter constructs including 5'-, 3'-fragment, and full length fragment from mouse were individually cloned into a luciferase reporter. Using dual-luciferase assay, the promoter harboring 3'-fragment contained much lower activity than the construct containing 5'-fragment which was though resistant to VPA induction, relative to 3'-fragment. Overexpression of Sp4 or Nrf2 increased whereas knockdown of either decreased Srr mRNA and SR protein. Using site-directed mutagenesis, mutation of Sp-binding elements or AREs in the constructs significantly decreased luciferase activity of the corresponding promoter construct. With chromatin immunoprecipitation, Sp4 was demonstrated to interact directly with the Sp-binding elements whereas Nrf2 bound AREs in Srr mRNA promoter. Altogether, our study highlights that Sp4 controls constitutive expression of SR in neuron and VPA mediates SR expression in N2A cells which is associated with its effect on neuron differentiation, that is, the effect is mediated via Nrf2.

Impairment of Store-operated Calcium Entry: Implications in Alzheimer's Neurodegeneration.

Alzheimer's disease (AD) is an insidious and progressive neurodegenerative disorder. Dysfunction of central cholinergic neurons, amyloid aggregation and deposition,oxidative stress,and biometal dyshomeostasis has been regarded as the major pathogenic mediators in this devastating disease. However, strategies derived from these hypotheses fail to slow down or stop the progression of AD, warranting a combination of therapies to target multiple etiological factors or examining alternative hypothesis. Store-operated calcium entry (SOCE) is the process by which depletion of calcium in the endoplasmic reticulum (ER) lumen causes an influx of calcium across plasmalemma. Accumulating evidence indicates that neuronal SOCE (nSOCE) is inhibited in family AD (FAD) and the inhibition of which causes instability of dendritic spines and enhances amyloidogenesis. Mutant Presenilin fails to function as an ER calcium leak channel and promotes degradation of stromal interaction molecules (STIM), ER calcium sensors; these effects may account for the repression of nSOCE in FAD. We have demonstrated that activation of autophagy degrades STIM proteins, resulting in a trimming effect on a dendritic arbor, under proteasome inhibition and endoplasmic reticulum stress, which are intimately connected with AD. Thus, we hypothesize that autophagy represses SOCE by degrading STIM proteins, leading to synapse loss in AD. This review article will highlight the roles of SOCE in AD neurodegeneration, the degradative mechanisms of STIM protein, and the therapeutic potential and associated challenge.

Autophagic degradation of stromal interaction molecule 2 mediates disruption of neuronal dendrites by endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress has been highlighted as one of the factors involved in axon/dendrite degeneration, which is an early event in Alzheimer's, Parkinson's diseases as well as in acute disorders such as ischemia and axotomy-induced retinal ganglion cell degeneration. These lines of evidence suggest critical roles of ER stress at the early stage of neurodegeneration, but the relevant mechanism is rarely exploited. In this study, we report that treatment with sublethal level of ER stressors, tunicamycin or brefeldin A, in primary rat neuronal cultures, significantly reduced dendrite arbor. Under the same treatment, either stressor reduced store-operated calcium entry (SOCE) and cytosolic calcium, [Ca2+ ]i , which were associated with autophagic degradation of stromal interaction molecule 2 (STIM2). Knockdown of ATG7 or activating transcription factor 4 completely reversed the reduction of STIM2 and significantly reversed the inhibition of SOCE under ER stress. Overexpression of STIM2 in neurons significantly prevented the ER stress-induced disruption of dendrite arbor. Altogether, our data reveal an unprecedented mechanism by which ER stress induces dendrite degeneration, that is, ER stress induces autophagic degradation of STIM2, leading to ensued SOCE inhibition and reduced [Ca2+ ]i , resulting in trimming effect on dendrites.

Intravenous injection of l-aspartic acid ß-hydroxamate attenuates choroidal neovascularization via anti-VEGF and anti-inflammation.

Choroidal neovascularization (CNV) is a hallmark of exudative age-related macular degeneration (exAMD) and a major cause of visual loss in AMD. Despite the widespread use of anti-VEGF therapy, serious adverse effects arise from repeated intravitreal injection of anti-VEGF antibodies, which warrant alternative strategy. We report herein that in a CNV murine model created by krypton red laser, intravenous injection of a serine racemase inhibitor, l-Aspartic acid ß-hydroxamate (L-ABH), significantly reduced CNV at the dose 6 mg/kg on the first day before and followed by 3 mg/kg on the third day after laser injury. The CNV volumes were analyzed with isolectin GS-IB4 staining on choroidal/RPE flat mounts on the seventh day after laser injury. Injection of L-ABH did not produce negative effects on retinal function and visual behavior. To dissect the mechanism in vitro, pretreatment with L-ABH in primary RPE cultures significantly reduced production of vascular endothelial growth factor (VEGF) and macrophage chemotactic protein 1 (MCP-1) by TNFα-primed RPEs. Consistent with these observations, L-ABH pretreatment significantly attenuated macrophage migration mediated by TNFα-primed RPE. Collectively, intravenous injection of L-ABH significantly reduced CNV volumes via reducing production of VEGF and MCP-1 by inflammation-primed RPEs.

MicroRNA-29 enhances autophagy and cleanses exogenous mutant αB-crystallin in retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPEs), a pigmented cell layer in the outer retina, are constantly exposed to photo-oxidative stress. Autophagy relieves the stress by removing oxidative protein adducts, protein aggregates, and damaged mitochondria. We previously found that miR-29 is downregulated in choroid/RPE tissue in a model of exudative age-related macular degeneration (AMD), suggesting that miR-29 deficiency may contribute to autophagy inhibition and AMD progression. Here we wanted to test whether overexpression of miR-29 in RPEs could enhance autophagy, thereby facilitating removal of drusen components. Indeed, overexpression of miR-29 in the RPEs increased autophagy, assessed by decreased protein levels of p62, increased lipid form of microtubule-associated protein light chain (LC3-II), and elevated autophagy flux. Furthermore, overexpression of miR-29 mitigated the formation of mutant αB-crystallin (R120G) protein aggregates. In probing the mechanism, we demonstrated that miR-29 post-transcriptionally repressed LAMPTOR1/p18 via targeting its 3'-UTRs of messenger RNA. MiR-29 overexpression and knockdown of LAMPTOR1/p18 led to limited mTORC1 recruitment to lysosomes and inhibition of mTORC1 activity. Altogether, miR-29 enhances autophagy which aids in removal of protein aggregates. These findings reveal a novel role of miR-29, which has the potential of being a therapeutic strategy for rescuing RPE degeneration in ocular disorders.

Loss-of-function mutation of serine racemase attenuates retinal ganglion cell loss in diabetic mice.

Consistent results suggest the promoting roles of serine racemase (SR)/D-serine in retinal neurodegeneration in diabetic retinopathy (DR). However, the direct evidence connecting SR deficiency with retinal neuroprotection in genetic model of diabetes mellitus has not been reported. In this investigation, we explore the effect of absence of functional SR on the degeneration of retinal ganglion cells (RGCs) with a diabetic murine model, Ins2Akita mice. We established a murine strain with double mutation, termed Ins2Akita-Srr, by mating heterozygous Ins2Akita mice with homozygous Srrochre269 mice. Ins2Akita retained less RGC in posterior, middle, and peripheral retinae than the counterpart from non-diabetic sibling mice at the age of five or seven months. Ins2Akita-Srr mice retained more RGC in middle and peripheral--but not in posterior-- retinae than the counterpart from Ins2Akita sibling mice at the age of five months. By contrast, at the age of seven months, Ins2Akita-Srr mice contained more RGC in peripheral, middle, and posterior retinae than the counterpart from Ins2Akita. RGCs were identified with retrograde labeling in vivo or with immunolabeling against a RGC-specific transcription factor, Brn3a, in retinal flat mounts. Correspondingly, the aqueous humor of Ins2Akita-Srr contained less amount of D-serine than sibling Ins2Akita mice. Thus, SR deficiency significantly prevented RGC loss in diabetic mice. We conclude that D-serine is a critical factor in the degeneration of RGC in DR. Targeting SR expression or activity may be a strategy for ameliorating RGC loss in DR.

Potential deficit from decreased cerebellar granule cell migration in serine racemase-deficient mice is reversed by increased expression of GluN2B and elevated levels of NMDAR agonists.

Inward migration of cerebellar granule cells (CGCs) after birth is critical for lamination in the cerebellar cortex. N-methyl-d-aspartate (NMDA) subtype of glutamate receptor (NMDAR) tethering CGCs into Bergmann glial fibers mediates the inward movement during the glial-dependent migratory phase. Activation of NMDAR depends on simultaneous binding of the GluN2 subunit by glutamate, and of the GluN1 subunit by d-serine or glycine; d-serine is believed to be an endogenous ligand of NMDAR. We hypothesized that lamination of the cerebellar cortex may be compromised in Srr (the gene for serine racemase (SR)) mutated mice (Srrnull) because of significantly low levels of d-serine per se. Indeed, the external germinal cell layer (EGL) in Srrnull was thicker than in sibling wild-type (WT) mice on postnatal day7 (P7), which accords with decreased CGC migration in Srrnull mice. However, the cerebellar laminar structure in Srrnull mice was normal on P12 and later. Feeding d-serine to pregnant mice abrogated the increased EGL thickness in Srrnull mice on P7. To determine the underlying mechanism of abnormal laminar structure during cerebellar development in Srrnull mice, we examined NMDAR subunits and their ligands. We found increased GluN2B on P10 and increased glycine during P7-12 in the cerebellar homogenates from Srrnull mice compared with the corresponding values from sibling WT mice. In summary, the study revealed how the potential defect in early cerebellar development caused by Srr mutation is circumvented by a compensatory mechanism. This knowledge advances understanding of the adaptation of cerebellar development under the condition of Srr mutation.

Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells.

Choroidal neovascularization (CNV) is a leading cause of blindness in age-related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d-serine, is an endogenous co-agonist of N-methyl-d-aspartate receptor. Activation of the receptor results in production of nitric oxide (. NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser-injured CNV mice, we found that inactivation of SR-coding gene (Srrnull ) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism in vivo and ex vivo. RPE from wild-type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser-injured Srrnull mice contained less inducible nitric oxide synthase and decreased phospho-VEGFR2 compared to amounts in WT mice. In vitro, inflammation-primed WT RPEs expressed more inducible NOS, produced more. NO and VEGF than did inflammation-primed Srrnull RPEs. When co-cultured with inflammation-primed Srrnull RPE, significantly fewer RF/6A-a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co-cultured with pre-treated WT RPE. Altogether, SR deficiency reduces RPE response to laser-induced inflammatory stimuli, resulting in decreased production of a cascade of pro-angiogenic cytokines, including. NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.

Disrupted-in-schizophrenia-1 as a broader link of glutamatergic transmission to schizophrenia impacts cerebral neurochemistry via the production of the 'gliotransmitter' d-serine, a NMDA receptor coagonist.

This Editorial highlights a study by Xia and coworkers in the current issue of the Journal of Neurochemistry, in which the authors reveal a possible mechanistic link between DISC1 (disrupted-in-schizophrenia-1), a genetic risk factor for schizophrenia, and N-methyl-d-aspartate receptor (NMDAR) that is also linked with schizophrenia. The authors show that perturbed communication between DISC1 and NMDARs represents a hidden perpetrator for abnormal dendritic and synaptic maturation. Read the highlighted article 'DISC1, astrocytes and neuronal maturation: a possible mechanistic link with implications for mental disorders' on page 518.

Inhibition of store-operated calcium entry by sub-lethal levels of proteasome inhibition is associated with STIM1/STIM2 degradation.

Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer's and Parkinson's diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer's disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG-132 and clasto-lactacystin-ß-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE.