Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism.

The histone demethylase KDM1A is a multi-faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A-/- hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a-/- hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells.

Rac1 and Rac3 GTPases differently influence the morphological maturation of dendritic spines in hippocampal neurons.

The Rac1 and Rac3 GTPases are co-expressed in the developing nervous system, where they are involved in different aspects of neuronal development, including the formation of synapses. The deletion of both Rac genes determines a stronger reduction of dendritic spines in vitro compared to the knockout of either gene, indicating that Rac1 and Rac3 play a synergistic role in the formation of these structures. Here, we have addressed the role of each GTPase in the formation of dendritic spines by overexpressing either Rac1 or Rac3 in wildtype neurons, or by re-expressing either GTPase in double knockout hippocampal cultures. We show that the Rac3 protein is expressed with Rac1 in developing hippocampal neurons. Overexpression of either GTPase in WT neurons increases the density of dendritic spines, suggesting the involvement of both GTPases in their formation. We also found that the re-expression of either Rac1 or Rac3 in double knockout neurons is sufficient to restore spinogenesis. Rac1 is significantly more efficient than Rac3 in restoring the formation of spines. On the other hand the quantitative analysis in neurons overexpressing or re-expressing either GTPase shows that Rac3 induces a more pronounced increase in the size of the spines compared to Rac1. These enlarged spines form morphological synapses identified by the juxtaposition of postsynaptic and presynaptic markers. Thus, while Rac1 appears more efficient in inducing the formation of mature spines, Rac3 is more efficient in promoting their enlargement. Our study highlights specific roles of Rac1 and Rac3, which may be functionally relevant also to synaptic plasticity.

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks.

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons.

Reduction in parvalbumin-positive interneurons and inhibitory input in the cortex of mice with experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS), inflammation leads to damage of central nervous system myelin and axons. Previous studies have postulated impaired GABA transmission in MS, and recent postmortem analysis has shown that GABAergic parvalbumin (PV)-positive interneurons are decreased in the primary motor cortex (M1) of patients with MS. In this report, we present evidence for the loss of a specific population of GABAergic interneurons in the experimental autoimmune encephalomyelitis mouse model of MS. Using experimental autoimmune encephalomyelitis, we evaluated the distribution of both PV-positive interneurons and of the inhibitory presynaptic input in the M1 of experimental autoimmune encephalomyelitis and control mice. Our results demonstrate a specific decrease in the number of PV-positive interneurons in the M1 of mice with experimental autoimmune encephalomyelitis. We detected a significant reduction in the number of PV-positive interneurons in the layers II and III of the M1 of diseased mice, while there was no difference in the number of calretinin (CR)-positive cells between animals with experimental autoimmune encephalomyelitis and control animals. Moreover, we observed a significant reduction in the inhibitory presynaptic input in the M1 of treated mice. These changes were specific for the mice with elevated clinical score, while they were not detectable in the mice with low clinical score. Our results support the hypothesis that reinforcing the action of the GABAergic network may represent a therapeutic alternative to limit the progression of the neuronal damage in MS patients.

Rac1 and rac3 GTPases control synergistically the development of cortical and hippocampal GABAergic interneurons.

The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.

Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Biochemical and functional characterisation of αPIX, a specific regulator of axonal and dendritic branching in hippocampal neurons.

BACKGROUND INFORMATION: PIX proteins are exchange factors for Rac and Cdc42 GTPases that are differentially expressed in the brain, where they are implicated in neuronal morphogenesis. The PIX family includes the two members αPIX and ßPIX, and the gene of αPIX is mutated in patients with intellectual disability. RESULTS: We have analysed the expression of PIX proteins in the developing brain and addressed their role during early hippocampal neuron development. Mass spectrometry identified several ßPIX isoforms and a major p75 αPIX isoform in brain and hippocampal cultures. PIX proteins expression increased with time during neuronal differentiation in vitro. The PIX partners GIT1 and GIT2 are also found in brain and their expression was increased during neuronal differentiation. We found that αPIX, but not ßPIX, was required for proper hippocampal neuron differentiation, since silencing of αPIX specifically hampered dendritogenesis and axonal branching. Interestingly, the depletion of GIT2 but not GIT1 mimicked the phenotype observed after αPIX knock-down. Over-expression of αPIX specifically enhanced dendritic branching, while both αPIX and ßPIX over-expression affected axonal morphology. Again, only over-expression of GIT2, but not GIT1, affected neuritic morphology. CONCLUSIONS: The results indicate that αPIX and GIT2 are required for neuronal differentiation, and suggest that they are part of the same pathway, while GIT1 and ßPIX are dispensable for early hippocampal neurons development.

Rac1 and Rac3 GTPases regulate the development of hilar mossy cells by affecting the migration of their precursors to the hilus.

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.

A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion.

BACKGROUND: Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. RESULTS: Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. CONCLUSION: Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

Expression of cyclin-dependent kinase 5 mRNA and protein in the human brain following acute ischemic stroke.

Neuronal cell death after brain ischemia may be regulated by activation of cyclin-dependent kinase 5 (Cdk5). In this study, expression of Cdk5 and its activator p35/p25 was examined in human post-mortem stroke tissue and in human cerebral cortical fetal neurons and human brain microvascular endothelial cells exposed to oxygen-glucose deficiency and reperfusion. The majority of patients demonstrated increased expression of Cdk5 and p-Cdk5 in stroke-affected tissue, with about a third showing increased p35 and p25 cleaved fragment as determined by Western blotting. An increase in Cdk5-, p-Cdk5- and p35-positive neurons and microvessels occurred in stroke-affected regions of patients. Staining of neurons became irregular and clumped in the cytoplasm, and nuclear translocation occurred, with colocalization of p35 and Cdk5. Association of Cdk5 with nuclear damage was demonstrated by coexpression of nuclear Cdk5 in TUNEL-positive neurons and microvessels in peri-infarcted regions. In vitro studies showed up-regulation and/or nuclear translocation of Cdk5, p-Cdk5 and p35 in neurons and endothelial cells subjected to oxygen-glucose deficiency, and strong staining was associated with propidium iodide positive nuclei, an indicator of cellular damage. These results provide new evidence for a role of Cdk5 in the events associated with response to ischemic injury in humans.

Cellular prion protein is increased in the plasma and peri-infarcted brain tissue after acute stroke.

The physiologic properties of the normal cellular prion protein (PrP(C)) have not been established fully, although recent evidence showed its upregulation in cerebral ischaemia. Using patients, animal models, and in vitro studies we aimed to identify in detail the expression and localization of PrP(C) in ischemic stroke. Patients in acute phase of ischaemic stroke had increased plasma levels of circulating PrP(C) as compared to healthy age- and gender-matched controls (3.1 +/- 1.4 vs. 1.9 +/- 0.7 ng/ml, P = 0.002). Immunohistochemistry showed increased expression of PrP(C) in the soma of peri-infarcted neurones as well as in the endothelial cells (EC) of micro-vessels and inflammatory cells in peri-infarcted brain tissue from patients who survived for 2-34 days after an initial stroke. The same pattern was repeated 1-48 hr after MCAO. RT-PCR showed increased gene expression of PrP(C) by human foetal neurons (HFN) after 12 hr of oxygen glucose deprivation (OGD), which remained increased after 24 hr reperfusion. Western blotting confirmed that protein expression was similarly upregulated, and fluorescent labeling showed a notable increase in peri-nuclear and axonal PrP(C) staining intensity. Increased plasma PrP(C) seems to reflect endogenous expression in acute stroke-affected brain tissue. Increased cellular expression in peri-infarcted regions may influence hypoxia-induced cell damage, although the effects on EC survival and angiogenesis remain to be elucidated.