Search

MEF2C has been shown to be a critical transcription factor for neurodevelopment, whose loss-of-function mutation in humans results in MEF2C haploinsufficiency syndrome (MHS), a severe form of autism spectrum disorder (ASD)/intellectual disability (ID). Here, we use patient hiPSC-derived cerebrocortical neurons and cerebral organoids to characterize MHS deficits. Unexpectedly, we found an aberrant micro-RNA-mediated gliogenesis pathway that contributes to decreased neurogenesis. We also demonstrate network-level hyperexcitability in neurons, as evidenced by excessive synaptic and extrasynaptic activity contributing to excitatory/inhibitory (E/I) imbalance. Notably, the extrasynaptic NMDA receptor antagonist, NitroSynapsin, corrects this aberrant electrical activity associated with abnormal phenotypes. During neurodevelopment, MEF2C regulates many ASD-associated gene networks suggesting that our approach may lead to personalized therapy for multiple forms of ASD.One sentence summaryAutism-like MEF2C+/- patient hiPSC models show miRNA-mediated overproduction of astrocytes and hyperactivity of neurons.

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/− mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/− phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/− mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.

HIV-associated neurocognitive disorder (HAND) consists of motor and cognitive dysfunction in a relatively large percentage of patients with AIDS. Prior work has suggested that at least part of the neuronal and synaptic damage observed in HAND may occur due to excessive stimulation of NMDA-type glutamate receptors (NMDARs). Here, we compared pharmacological and genetic manipulation of NMDAR activity using an improved derivative of the NMDAR antagonist memantine, termed NitroMemantine, and the modulatory NMDAR subunit GluN3A in the HIV/gp120 transgenic (tg) mouse model of HAND. Interestingly, we found that while both NitroMemantine and GluN3A have been shown to inhibit NMDAR activity, NitroMemantine protected synapses in gp120 tg mice, but overexpression of GluN3A augmented the damage. Given recent findings in the field, one explanation for this apparently paradoxical result is the location of the NMDARs primarily affected, with NitroMemantine inhibiting predominantly extrasynaptic pathologically-activated NMDARs, but GluN3A disrupting normal NMDAR-mediated neuroprotective activity via inhibition of synaptic NMDARs.

[Briefly describe the contents of the Data in Brief article. Tell the reader the repository and reference number for the data in the abstract to.] The myocyte enhancer factor 2 (MEF2) family of transcription factors is highly expressed in the brain, and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs) derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA), which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184). By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were upregulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c +/−(Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention., Human MEF2C haploinsufficiency results in Autism Spectrum Disorder (ASD), but it is unclear if the same is true in mice. Here, the authors show that Mef2c +/−mice have behavioral defects and neuronal abnormalities similar to ASD, and symptoms can be ameliorated with the new drug, NitroSynapsin.

Purpose Photoreceptor degeneration in the retina is a major cause of blindness in humans. Elucidating mechanisms of degenerative and neuroprotective pathways in photoreceptors should afford identification and development of therapeutic strategies. Methods We used mouse genetic models and improved methods for retinal explant cultures. Retinas were enucleated from Mef2d+/+ and Mef2d-/- mice, stained for MEF2 proteins and outer nuclear layer thickness, and assayed for apoptotic cells. Chromatin immunoprecipitation (ChIP) assays revealed MEF2 binding, and RT-qPCR showed levels of transcription factors. We used AAV2 and electroporation to express genes in retinal explants and electroretinograms to assess photoreceptor functionality. Results We identify a prosurvival MEF2D-PGC1α pathway that plays a neuroprotective role in photoreceptors. We demonstrate that Mef2d-/- mouse retinas manifest decreased expression of PGC1α and increased photoreceptor cell loss, resulting in the absence of light responses. Molecular repletion of PGC1α protects Mef2d-/- photoreceptors and preserves light responsivity. Conclusions These results suggest that the MEF2-PGC1α cascade may represent a new therapeutic target for drugs designed to protect photoreceptors from developmental- and age-dependent loss.

Gaining mechanistic insight into interaction between causative factors of complex multifactorial diseases involving photoreceptor damage might aid in devising effective therapies. Oxidative stress is one of the potential unifying mechanisms for interplay between genetic and environmental factors that contribute to photoreceptor pathology. Interestingly, the transcription factor myocyte enhancer factor 2d (MEF2D) is known to be important in photoreceptor survival, as knockout of this transcription factor results in loss of photoreceptors in mice. Here, using a mild light-induced retinal degeneration model, we show that the diminished MEF2D transcriptional activity in Mef2d+/- retina is further reduced under photostimulation-induced oxidative stress. Reactive oxygen species cause an aberrant redox modification on MEF2D, consequently inhibiting transcription of its downstream target, nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2 is a master regulator of phase II antiinflammatory and antioxidant gene expression. In the Mef2d heterozygous mouse retina, NRF2 is not up-regulated to a normal degree in the face of light-induced oxidative stress, contributing to accelerated photoreceptor cell death. Furthermore, to combat this injury, we found that activation of the endogenous NRF2 pathway using proelectrophilic drugs rescues photoreceptors from photo-induced oxidative stress and may therefore represent a viable treatment for oxidative stress-induced photoreceptor degeneration, which is thought to contribute to some forms of retinitis pigmentosa and age-related macular degeneration.

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not.In vivo, cerebella inMef2dknock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT.

Significance S -nitrosylation, addition of an NO group to a cysteine thiol, can regulate protein activity. Aberrant protein S -nitrosylation, however, can disrupt normal enzyme function, as is the case for S -nitrosylated peroxiredoxin (SNO-Prx), which would otherwise catabolize toxic peroxides that occur under neurodegenerative conditions such as Parkinson’s disease. Here, we describe a paradigm of N -phosphorylation–mediated denitrosylation by the enzyme sulfiredoxin that removes NO from Prx. The findings are at the center of redox control of the cell, explaining reactivation by sulfiredoxin of both Prx-SO 2 H and SNO-Prx and thus describe a master regulator of redox reactions that combats nitrosative and oxidative stress in cells. These results suggest that sulfiredoxin may be an important target for therapeutic intervention in neurodegenerative disorders.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in 'Big Pharma,' 'undruggable' for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic N-Methyl-D-aspartic acid receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc to contribute to emotional and motivational learning. In an attempt to identify presynaptically-located NMDARs in the NAc, the present study utilizes slice electrophysiological recording combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid (DCKA) or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of D-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.

NMDA receptors are typically excited by a combination of glutamate and glycine. Here we describe excitatory responses in CNS myelin that are gated by a glycine agonist alone and mediated by NR1/NR3 “NMDA” receptor subunits. Response properties include activation byd-serine, inhibition by the glycine-site antagonist CNQX, and insensitivity to the glutamate-site antagonistd-APV.d-Serine responses were abrogated in NR3A-deficient mice. Our results suggest the presence of functional NR1/NR3 receptors in CNS myelin.

Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo , resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-d-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extrasynaptic receptors, likely composed of NR1, NR2, and NR3 subunits.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in ‘Big Pharma,’ ‘undruggable’ for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

Hitherto, membralin has been a protein of unknown function. Here, we show that membralin mutant mice manifest a severe and early-onset motor neuron disease in an autosomal recessive manner, dying by postnatal day 5–6. Selective death of lower motor neurons, including those innervating the limbs, intercostal muscles, and diaphragm, is predominantly responsible for this fatal phenotype. Neural expression of a membralin transgene completely rescues membralin mutant mice. Mechanistically, we show that membralin interacts with Erlin2, an endoplasmic reticulum (ER) membrane protein that is located in lipid rafts and known to be important in ER-associated protein degradation (ERAD). Accordingly, the degradation rate of ERAD substrates is attenuated in cells lacking membralin. Membralin mutations or deficiency in mouse models induces ER stress, rendering neurons more vulnerable to cell death. Our study reveals a critical role of membralin in motor neuron survival and suggests a novel mechanism for early-onset motor neuron disease. DOI: http://dx.doi.org/10.7554/eLife.06500.001, eLife digest As new proteins are built inside a cell, many will pass into a structure called the endoplasmic reticulum for processing. There, the proteins are folded into the specific three-dimensional shapes that allow them to carry out their respective jobs. Sometimes the folding process goes awry, leading to a build-up of unfolded proteins that stress the endoplasmic reticulum and can kill the cell. Brain cells are particularly vulnerable to death from endoplasmic reticulum stress. To combat a deadly build-up of unfolded proteins, each cell has systems that respond when the endoplasmic reticulum is under stress. Unchecked stress on the endoplasmic reticulum has been linked to diseases like amyotrophic lateral sclerosis (called ALS for short). In diseases like ALS, the nerve cells that control muscle movements gradually die off, causing a loss of muscle control and eventually death. Scientists suspect that these nerve cells (called motor neurons) are particularly sensitive to endoplasmic reticulum stress because they are highly active. Drugs that help counteract stress on the endoplasmic reticulum extend the lives of mice with motor neuron disease, suggesting this may be a useful strategy for treating such diseases in humans. Now, Yang, Qu et al. identify a new protein that appears necessary for a healthy endoplasmic reticulum. Mice that lack the gene for a protein called membralin die within five or six days after birth because their motor neurons die off. Further experiments showed that re-introducing membralin in their nervous system can rescue these membralin-deficient mice. Yang, Qu et al. found that membralin interacts with another protein that helps eliminate poorly folded or unfolded proteins in the endoplasmic reticulum, and thus relieves stress on the cell. Mutations in this endoplasmic reticulum stress response protein have previously been linked to motor neuron diseases. The motor neurons in membralin-deficient mice show signs of endoplasmic reticulum stress and are extra vulnerable to chemicals that induce protein misfolding. Together, the experiments show membralin plays an important role in mitigating stress on the endoplasmic reticulum. More studies of mice lacking membralin may help explain why the endoplasmic reticulum stress increases in motor neuron diseases and may point to possible treatments. DOI: http://dx.doi.org/10.7554/eLife.06500.002

Background The N-methyl-D-aspartate (NMDA) receptor is composed of various conformations of multiple subunits (including NR1, NR2A-D, and NR3A-B). Peak expression of the NR3A subunit occurs approximately 2–3 weeks postnatal, with low levels in adulthood. In the brain, the NR3A subunit is localized primarily in the amygdala, hippocampus, striatum, and cortex. These regions are involved in the modulation of prepulse inhibition of startle (PPI), an operational measure of sensorimotor gating that is modulated by NMDA receptors. NR3A reduces NMDA current in native neurons expressing NR1 and NR2 subunits and forms glycine receptors when expressed with NR1 in the absence of NR2 in both oocyte and mammalian expression systems. Methods To examine the role of NR3A in vivo, NR3A knockout (KO), and overexpressing transgenic mice were generated. Adult NR3A overexpressing mice exhibited normal PPI; PPI in NR3A KO mice was tested repeatedly from weaning through adulthood. Results Male NR3A KO mice exhibited an increase in PPI at 3 and 4 weeks postnatal, whereas female NR3A KO mice did not differ from their WT counterparts at any age tested. Conclusions This sex-specific increase in PPI is consistent with the antagonistic role of the NR3A subunit in NMDA receptor function and with the observation that estrogen modulates NMDA receptor function.

Alzheimer’s disease (AD) is characterized by synaptic and neuronal loss, which occurs at least partially through oxidative stress induced by oligomeric amyloid-β (Aβ)-peptide. Carnosic acid (CA), a chemical found in rosemary and sage, is a pro-electrophilic compound that is converted to its active form by oxidative stress. The active form stimulates the Keap1/Nrf2 transcriptional pathway and thus production of phase 2 antioxidant enzymes. We used both in vitro and in vivo models. For in vitro studies, we evaluated protective effects of CA on primary neurons exposed to oligomeric Aβ. For in vivo studies, we used two transgenic mouse models of AD, human amyloid precursor protein (hAPP)-J20 mice and triple transgenic (3xTg AD) mice. We treated these mice trans-nasally with CA twice weekly for 3 months. Subsequently, we performed neurobehavioral tests and quantitative immunohistochemistry to assess effects on AD-related phenotypes, including learning and memory, and synaptic damage. In vitro, CA reduced dendritic spine loss in rat neurons exposed to oligomeric Aβ. In vivo, CA treatment of hAPP-J20 mice improved learning and memory in the Morris water maze test. Histologically, CA increased dendritic and synaptic markers, and decreased astrogliosis, Aβ plaque number, and phospho-tau staining in the hippocampus. We conclude that CA exhibits therapeutic benefits in rodent AD models and since the FDA has placed CA on the ‘generally regarded as safe’ (GRAS) list, thus obviating the need for safety studies, human clinical trials will be greatly expedited.

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD50 for exposure to glutamate (10(-3)-10(-5) M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.

The synaptic toxicity of soluble amyloid-β (Aβ) oligomers plays a critical role in the pathophysiology of Alzheimer's disease (AD). Here we report that overexpressed α1-takusan, which we previously identified as a protein that enhances synaptic activity via interaction with PSD-95, mitigates oligomeric Aβ-induced synaptic loss. In contrast, takusan knockdown results in enhanced synaptic damage. α1-Takusan interacts with tau either directly or indirectly, and prevents Aβ-induced tau hyperphosphorylation and mitochondrial fragmentation. Deletion analysis identified the second domain (D2) within the takusan protein that is required for PSD-95 clustering and synaptic protection from Aβ. A 51 aa sequence linking D2 to the PDZ-binding C terminus was found to be as effective as full-length takusan in protecting synapses from Aβ-induced damage. Moreover, a sequence containing the D2 from the human protein discs large homolog 5, when linked to a C-terminal PDZ-binding motif, can also increase the clustering of PSD-95 in cortical dendrites. In summary, α1-takusan protects synapses from Aβ-induced insult via interaction with PSD-95 and tau. Thus, takusan-based protein sequences from either mouse or human may be of potential therapeutic benefit in AD.

Background Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson’s disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. Results We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. Conclusions Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.

Huntington's disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntington's disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression.

Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.

Parkinson’s disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD., Parkinson Society Canada (Fellowship), United Mitochondrial Disease Foundation (grant), National Institutes of Health (U.S.) (NIH grant P01 HD29587), National Institutes of Health (U.S.) (NIH grant P01 ES016738), National Institutes of Health (U.S.) (NIH grant P30 NS076411), National Institutes of Health (U.S.) (NIH grant R37 CA084198)

Hyperactivation of NMDA-type glutamate receptors (NMDARs) results in excitotoxicity, contributing to damage in stroke and neurodegenerative disorders. NMDARs are generally comprised of NR1/NR2 subunits but may contain modulatory NR3 subunits. Inclusion of NR3 subunits reduces the amplitude and dramatically decreases the Ca2+permeability of NMDAR-associated channels in heterologous expression systems and in transgenic mice. Since excessive Ca2+influx into neurons is a crucial step for excitotoxicity, we asked whether NR3A subunits are neuroprotective. To address this question, we subjected neurons genetically lacking NR3A to various forms of excitotoxic insult. We found that cultured neurons prepared from NR3A knock-out (KO) mice displayed greater sensitivity to damage by NMDA application than wild-type (WT) neurons.In vivo, neonatal, but not adult, WT mice contain NR3A in the cortex, and neonatal NR3A KO mice manifested more damage than WT after hypoxia–ischemia. In adult retina, one location where high levels of NR3A normally persist into adulthood, injection of NMDA into the eye killed more retinal ganglion cells in adult NR3A KO than WT mice. These data suggest that endogenous NR3A is neuroprotective. We next asked whether we could decrease excitotoxicity by overexpressing NR3A. We found that cultured neurons expressing transgenic (TG) NR3A displayed greater resistance to NMDA-mediated neurotoxicity than WT neurons. Similarlyin vivo, adult NR3A TG mice subjected to focal cerebral ischemia manifested less damage than WT mice. These data suggest that endogenous NR3A protects neurons, and exogenously added NR3A increases neuroprotection and could be potentially exploited as a therapeutic.

NR3A is a developmentally regulated N-methyl-D-aspartate receptor (NMDAR) subunit that was previously known as NMDAR-L or chi-1. Unlike other NMDAR subunits, NR3A inhibits the NMDAR-associated ion channel in a novel manner, and a role in synaptogenesis has been suggested for this subunit. Here, we report a comprehensive study to delineate the temporal and anatomic expression of NR3A protein in the mammalian brain by using a monoclonal anti-NR3A antibody. NR3A protein was found to peak at postnatal day (P) 8, and to decrease gradually from P12 to adulthood in the rat central nervous system. Moreover, NR3A protein was heavily expressed in all areas of the isocortex, portions of the amygdaloid nuclei, and selective cell layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem, and spinal cord. NR3A protein was also expressed in the cerebellar cortex, whereas only weak signal was detected in the previous in situ studies by using riboprobes. At an ultrastructural level, NR3A was associated specifically with asymmetrical synapses and localized to postsynaptic membranes. This information will facilitate future research on NMDARs by providing clues to possible inclusion of the NR3A subunit in NMDARs in many brain regions.

Recently, we cloned and began to characterize a new N-methyl-d-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca2+ permeability, decrease in Mg2+ sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8( P8) and similarly found to be relatively insensitive to Mg2+ block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.

Protein kinase inhibitor (PKI) is an endogenous inhibitor of cAMP-dependent protein kinase A (PKA). We have found that the alpha-isoform of PKI (PKIalpha) is asymmetrically expressed along the left-right (L-R) axis in chick embryos. At stage 6, PKIalpha is expressed on the right side of the node, and this asymmetric expression continues until stage 7+. After stage 8, PKIalpha expression returns symmetric. Treatment of embryos with antisense PKIalpha oligonucleotides increased the incidence of reversed heart looping. Antisense oligonucleotides also induced ectopic expression of the left-specific genes Nodal and Pitx2, and suppressed the expression of the right-specific gene SnR in the right lateral plate mesoderm. Similarly, treatment with PKA activators forskolin and Sp-cAMPs resulted in both reversed heart looping and bilateral expression of NODAL: Ectopic activin induced PKIalpha on the left side of the node, while ectopic Shh and anti-Shh antibody had no effect on PKIalpha expression. Taken together, these data suggest that PKIalpha induced by an activin-like molecule, through the inhibition of PKA activity, suppresses the Nodal-Pitx2 pathway on the right side of the body.

The NMDA (N -methyl-D-aspartate) subclass of glutamate receptor1 is essential for the synaptic plasticity thought to underlie learning and memory2,3,4 and for synaptic refinement during development5,6. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit7,8,9,10,11. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or χ-1), which is expressed primarily during brain development12,13. NR3Aco-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.

We have used expression cloning in Xenopus oocytes to isolate two different cDNAs encoding functional N-methyl-D-aspartate (NMDA) receptor subunits. The two receptors (NMDA-R1A and -R1B) display different pharmacologic properties as a consequence of alternative exon addition within the putative ligand-binding domain. The splicing choice is regulated such that R1B is the predominant form of receptor in the cerebellum, whereas R1A predominates in other brain regions. Expression of either of the subunits alone in oocytes results in an NMDA-evoked inward current with electrophysiologic properties closely resembling those of the NMDA receptors observed in neurons. Thus, the complex properties exhibited by the NMDA receptor in neurons can be generated by the expression of a single receptor subunit.

In their Research Article (“Identifying autism loci and genes by tracing recent shared ancestry,” 11 July 2008, p. [218][1]), E. M. Morrow et al. showed that gene expression associated with autism-spectrum disorders (ASD) is controlled by MEF2 transcription factors and hypothesized that autistic

Too much or too little NMDA receptor activity during development can result in neuronal cell death. Recent work suggests that NMDA receptor blockade can cause excessive neuronal apoptosis in the perinatal brain.

PURPOSE: Recently, a novel N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A, has been discovered in the brain and shown to decrease NMDAR activity by modulating the calcium permeability of the receptor channel. The insertion of NR3A within the NMDAR complex may thus alter NMDAR properties and play a crucial role during processes of neuronal development and degeneration. The present study is the first to investigate the expression and cellular localization of NR3A on the protein level in the retina and to elucidate its putative functional roles within the retinal circuitry. METHODS: The expression of NR3A in the retina was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. Functional aspects of NR3A in the retina were addressed by measuring the NMDA-induced increase in intracellular calcium, [Ca(2+)](i), in retinal cells prepared from wild-type (NR3A(+/+)) and NR3A knockout (NR3A(+/-), and NR3A(-/-)) mice. RESULTS: NR3A protein expression was initially observed in the first postnatal week and was predominantly localized to cell bodies in the ganglion cell layer. In older animals, two bands of NR3A immunoreactivity were additionally observed in the inner plexiform layer. NMDA-evoked [Ca(2+)](i) responses were found to be significantly greater in retinal cells in NR3A(-/-) mice than in wild-type retinas. CONCLUSIONS: The data indicate that NR3A is specifically expressed in the inner retina and may modulate NMDAR-mediated calcium influx and thus [Ca(2+)](i) levels in retinal ganglion cells and amacrine cells.

By selecting activities to complement Escherichia coli aro mutations, a gene responsible for the biosynthesis of aromatic amino acids in Schizosaccharomyces pombe (aro3) was cloned into pBR322. Three independent clones named pFNA1, pFNA2 and pFNA3 were obtained. pFNA1 could complement E. coli aroD only, whereas the other two plasmids were able to complement both aroD and aroE. The aro3 locus of S. pombe was found to be a gene cluster which can be subdivided into five complons, A through E (Strauss 1979). Transformation of S. pombe mutants defective in each complon with the pFNA plasmids indicated that pFNA1 carries at least a part of aro3A, the whole of B, C and D, as well as a part of E, whereas pFNA2 and pFNA3 carry a part of aro3C and the entire D and E complons. A physical map of the aro3 locus was constructed by analyzing the structure of these plasmids and their hybridization patterns to restricted genomic DNA. A transcript 4.5 kb long was detected as the sole aro3 mRNA encompassing all five complons. This study, in addition to the work of Strauss (1979), establishes that the arrangement of the aro3 complons in S. pombe in terms of their enzymatic coding activities is identical with that of Neurospora crassa, but different from that proposed for Saccharomyces cerevisiae.