Zinc accumulation in heterozygous mutants of fumble, the pantothenate kinase homologue of Drosophila

Coenzyme A (CoA) functions in the intracellular trafficking of acetyl groups. In humans, mutations in the pantothenate kinase-2 gene, which encodes a key enzyme in CoA biosynthesis, are associated with neurodegeneration and premature death. Diagnosis is based on iron accumulation in the globus pallidus observed by magnetic resonance imaging. We investigated the elemental composition of the fumble mutant, a model of the human disease. Surprisingly, flies carrying a fumble loss-of-function allele had a three-fold increase in total zinc levels per dry weight when compared to control strains, but no change in total iron, copper or manganese levels. Accordingly, zinc supplementation had an adverse impact on the development of fumble mutant larvae, but zinc chelation failed to protect.     

Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

Background

Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.

Objective, Methods, Results and Conclusions

Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.     

Acanthocytosis and the c.680 A>G Mutation in the PANK2 Gene: A Study Enrolling a Cohort of PKAN Patients from the Dominican Republic

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). Pantothenate kinases catalyze the rate-limiting step of coenzyme A synthesis and Pank2 is the only pantothenate kinase isoform in humans that is localized to mitochondria. Acanthocytosis, the occurrence of spiculated erythrocytes, is observed in about 10% of the PKAN patients. Therefore PKAN is also classified together with other rare neurodegenerative diseases like Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) into the Neuroacanthocytosis (NA) syndromes. It has not been investigated yet whether acanthocytosis in PKAN is associated with a specific subset of Pank2 mutations. In this study, we analyzed acanthocytosis of a cohort of 25 PKAN patients from the Dominican Republic that are homozygous for the c.680 A>G mutation in the PANK2 gene as compared to control donors that are heterozygous or wild-type with respect to this mutation. 3D modeling of this mutation indicated that the replacement of a tyrosine by a cysteine at position 227 in Pank2 disrupts a polar interaction within the A domain of the enzyme. Mean acanthocyte count was elevated in the cohort of patients, however, acanthocytosis varied among the patients with nearly half of them showing high (>20%) or elevated acanthocytosis and the rest showing mild (6-10%) or no (<6%) acanthocytosis. Heterozygous control donors revealed a tendency to mild acanthocytosis. Based on the insight that Pank2 is a normal constituent of red blood cells and de novo biosynthesis of coenzyme A is likely to take place in the erythrocyte cytosol we propose a hypothetical model that accounts for the variability in the occurrence of acanthocytic cells in PKAN.     

Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.     

GABAergic interneuron to astrocyte signalling: a neglected form of cell communication in the brain

GABAergic interneurons represent a minority of all cortical neurons and yet they efficiently control neural network activities in all brain areas. In parallel, glial cell astrocytes exert a broad control of brain tissue homeostasis and metabolism, modulate synaptic transmission and contribute to brain information processing in a dynamic interaction with neurons that is finely regulated in time and space. As most studies have focused on glutamatergic neurons and excitatory transmission, our knowledge of functional interactions between GABAergic interneurons and astrocytes is largely defective. Here, we critically discuss the currently available literature that hints at a potential relevance of this specific signalling in brain function. Astrocytes can respond to GABA through different mechanisms that include GABA receptors and transporters. GABA-activated astrocytes can, in turn, modulate local neuronal activity by releasing gliotransmitters including glutamate and ATP. In addition, astrocyte activation by different signals can modulate GABAergic neurotransmission. Full clarification of the reciprocal signalling between different GABAergic interneurons and astrocytes will improve our understanding of brain network complexity and has the potential to unveil novel therapeutic strategies for brain disorders.     

Mitochondria: A crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of brain iron deposition syndromes that lead to mixed extrapyramidal features and progressive dementia. Exact pathologic mechanism of iron deposition in NBIA remains unknown. However, it is becoming increasingly evident that many neurodegenerative diseases are hallmarked by metabolic dysfunction that often involves altered lipid profile. Among the identified disease genes, four encode for proteins localized in mitochondria, which are directly or indirectly implicated in lipid metabolism: PANK2, CoASY, PLA2G6 and C19orf12. Mutations in PANK2 and CoASY, both implicated in CoA biosynthesis that acts as a fatty acyl carrier, lead, respectively, to PKAN and CoPAN forms of NBIA. Mutations in PLA2G6, which plays a key role in the biosynthesis and remodeling of membrane phospholipids including cardiolipin, lead to PLAN. Mutations in C19orf12 lead to MPAN, a syndrome similar to that caused by mutations in PANK2 and PLA2G6. Although the function of C19orf12 is largely unknown, experimental data suggest its implication in mitochondrial homeostasis and lipid metabolism. Altogether, the identified mutated proteins localized in mitochondria and associated with different NBIA forms support the concept that dysfunctions in mitochondria and lipid metabolism play a crucial role in the pathogenesis of NBIA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Absence of an orphan mitochondrial protein, c19orf12, causes a distinct clinical subtype of neurodegeneration with brain iron accumulation

The disease classification neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive neurodegenerative disorders characterized by brain iron deposits in the basal ganglia. For about half of the cases, the molecular basis is currently unknown. We used homozygosity mapping followed by candidate gene sequencing to identify a homozygous 11 bp deletion in the orphan gene C19orf12. Mutation screening of 23 ideopathic NBIA index cases revealed two mutated alleles in 18 of them, and one loss-of-function mutation is the most prevalent. We also identified compound heterozygous missense mutations in a case initially diagnosed with Parkinson disease at age 49. Psychiatric signs, optic atrophy, and motor axonal neuropathy were common findings. Compared to the most prevalent NBIA subtype, pantothenate kinase associated neurodegeneration (PKAN), individuals with two C19orf12 mutations were older at age of onset and the disease progressed more slowly. A polyclonal antibody against the predicted membrane spanning protein showed a mitochondrial localization. A histopathological examination in a single autopsy case detected Lewy bodies, tangles, spheroids, and tau pathology. The mitochondrial localization together with the immunohistopathological findings suggests a pathomechanistic overlap with common forms of neurodegenerative disorders.     

d-Serine and Serine Racemase are Localized to Neurons in the Adult Mouse and Human Forebrain

d-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from l-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, d-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR?/?) mice, which have <15 % of normal d-serine levels, to validate and optimize a d-serine immunohistochemical method. Nearly all of the d-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no d-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the d-serine positive neurons contained SR in the neocortex and HP. Greater than half of the d-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25–40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100β-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of d-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of d-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.     

Differential roles of alanine in GABAergic and glutamatergic neurons

Studies in different preparations of neurons and astrocytes of alanine transport and activities of its metabolizing enzyme alanine aminotransferase have led to the proposal that this amino acid is preferentially synthesized in astrocytes and transferred from the astrocytic to the neuronal compartment. From a functional point of view this may well be the case in a GABAergic synapse since theoretically alanine can be utilized as a metabolic fuel in GABAergic neurons where the GABA shunt is operating. Thus, a metabolic scheme is proposed, according to which alanine catabolism is coupled to the TCA cycle where the GABA shunt replaces the alpha-ketoglutarate dehydrogenase/succinyl CoA synthetase reactions. In a glutamatergic synapse in which the large demand for synthesis of neurotransmitter glutamate leads to a large production of ammonia, it is possible that alanine could play a completely different role. Hence, experimental evidence is reviewed suggesting that alanine may serve as a carrier of ammonia nitrogen from the neuronal compartment to the astrocytic compartment using a flux of lactate in the opposite direction to account for transfer of the C-3 carbon skeleton.     

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25^+/− mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.     

The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis

Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is essential for growth of the intraerythrocytic stage of human and avian malaria parasites. Avian malaria parasites have been reported to be incapable of de novo CoA synthesis and instead salvage CoA from the host erythrocyte; hence, pantothenate is required for CoA biosynthesis within the host cell and not the parasite itself. Whether the same is true of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, remained to be established. In this study we investigated the metabolic fate of [¹⁴C]pantothenate within uninfected and P. falciparum-infected human erythrocytes. We provide evidence consistent with normal human erythrocytes, unlike rat erythrocytes (which have been reported to possess an incomplete CoA biosynthesis pathway), being capable of CoA biosynthesis from pantothenate. We also show that CoA biosynthesis is substantially higher in P. falciparum-infected erythrocytes and that P. falciparum, unlike its avian counterpart, generates most of the CoA synthesized in the infected erythrocyte, presumably necessitated by insufficient CoA biosynthesis in the host erythrocyte. Our data raise the possibility that malaria parasites rationalize their biosynthetic activity depending on the capacity of their host cell to synthesize the metabolites they require.Pantothenate (vitamin B₅) is an essential nutrient for the virulent human malaria parasite Plasmodium falciparum, required to support the rapid growth and replication of the parasite during the intraerythrocytic stage of its life cycle (1–3). In bacteria, plants, and mammalian tissues, pantothenate serves as a precursor of coenzyme A (CoA),³ an essential enzyme cofactor involved in numerous metabolic reactions in the cell. Pantothenate is converted to CoA via five universal enzyme-mediated steps (see Fig. 1).Open in a separate windowFIGURE 1.The CoA biosynthesis pathway.Several decades ago, Trager (4) showed that pantothenate supported the survival of the avian malaria parasite Plasmodium lophurae during its development within duck erythrocytes in vitro. Trager (5, 6) later demonstrated, however, that CoA, and not pantothenate, stimulated exoerythrocytic growth of the intraerythrocytic stage of P. lophurae, and proposed that avian malaria parasites are incapable of metabolizing pantothenate to CoA, and instead rely on CoA synthesized by the host erythrocyte. In support of this proposal, CoA biosynthesis enzymes are readily detectable in duck erythrocytes, but appear to be absent from P. lophurae parasites isolated from their host erythrocyte (7, 8). Pantothenate is therefore required by the P. lophurae-infected duck erythrocyte for CoA biosynthesis within the host cell and not the parasite itself.By contrast with nucleated avian erythrocytes, mammalian erythrocytes are thought to be incapable of CoA biosynthesis. In the only study on the subject, Annous and Song (9) reported that although pantothenate is phosphorylated within rat erythrocytes (the first step in CoA biosynthesis), there is no evidence for the subsequent steps of the CoA biosynthesis pathway. Saliba et al. (10) confirmed that human erythrocytes similarly phosphorylate pantothenate, but did not investigate whether CoA synthesis also occurs in the cells. A lack of CoA biosynthesis in mammalian erythrocytes would seemingly place the burden of CoA synthesis squarely on malaria parasites that infect mammals (such as P. falciparum), contrary to the situation in birds. Although Saliba et al. (10) have shown that P. falciparum is capable of performing the first step in CoA biosynthesis, it remains to be established whether the parasite can metabolize the 4′-phosphopantothenate generated from pantothenate to CoA or, like P. lophurae, relies on CoA synthesized in the host erythrocyte for its normal growth and replication.In this study we followed the metabolism of pantothenate within uninfected human erythrocytes, P. falciparum-infected human erythrocytes, and isolated P. falciparum parasites. We provide evidence that both uninfected erythrocytes (which we show do take up pantothenate, albeit very slowly) and P. falciparum-infected erythrocytes synthesize CoA from pantothenate. CoA biosynthesis is, however, dramatically higher in the P. falciparum-infected cell. Furthermore, we show that P. falciparum parasites synthesize CoA in the absence of the host erythrocyte, and hence, by contrast with avian malaria parasites, the human malaria parasite does not rely on the host erythrocyte for CoA.     

Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.     

Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.     

Astrocytes are important mediators of A��-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer''s disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

Astroglial connexin 43 sustains glutamatergic synaptic efficacy

Astrocytes dynamic interactions with neurons play an active role in neurotransmission. The gap junction (GJ) subunits connexins 43 and 30 are strongly expressed in astrocytes and have recently been shown to regulate synaptic activity and plasticity. However, the specific role of connexin 43 in the morphological and electrophysiological properties of astrocytes in situ as well as in synaptic transmission remains unknown. Here, we show that connexin 43, a major determinant of astroglial GJ coupling, regulates astrocyte cell volume, but has no impact on astroglial passive membrane properties. Furthermore, we demonstrate that connexin 43 modulates glutamatergic synaptic activity of hippocampal CA1 pyramidal cells. This regulation involves changes in synaptically released glutamate, with no alteration in neuronal excitability or postsynaptic function. These results reveal connexin 43 as a critical player in neuroglial interactions by supporting synaptic efficacy.     

Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.