Summary Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular and electrophysiological analysis we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development and synapse formation in layer II–III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2−/− mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight novel subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits.

Whereas neurons of the lower layers (VI-V) of the cerebral cortex are first born from dividing precursors at the ventricular zone, upper layer neurons (II-IV) subsequently arise from divisions of intermediate neuronal precursors at the subventricular zone (SVZ). Little is known about mechanisms that control the proliferation of SVZ neuronal precursors. We herein report that the restricted expression of the homeodomain transcription factor Cux-2 in the SVZ regulates the proliferation of intermediate neuronal precursors and the number of upper layer neurons. In Cux-2-deficient mice (Cux-2-/-), there is excessive number of upper layer neurons and selective expansion of SVZ neuronal precursors. Double-labeling experiments demonstrate that Cux-2-/- upper layer precursors reenter the cell cycle in a higher frequency than wild-type precursors. Overexpression studies indicate that Cux-2 controls cell cycle exit in a cell-autonomous manner. Analysis of Cux-1-/-; Cux-2-/- double mutant revealed that Cux-2 controls SVZ proliferation independently of Cux-1, demonstrating that this is a unique function of Cux-2, not redundant with Cux-1 activities. Our results point to Cux-2 as a key element in the control of the proliferation rates of the SVZ precursors and the number of upper cortical neurons, without altering the number of deep cortical layers.

The proposed presence of P2X7 receptor (P2X7R) in neurons has been the source of some contention. Initial studies suggested an absence of P2X7R mRNA in neurons, and the apparent nonspecificity of the antibodies used to identify P2X7R raised further doubts. However, subsequent studies using new pharmacological and biomolecular tools provided conclusive evidence supporting the existence of functional P2X7Rs in neurons. The P2X7 receptor has since been shown to play a leading role in multiple aspects of neuronal physiology, including axonal elongation and branching and neurotransmitter release. P2X7R has also been implicated in neuronal pathologies, in which it may influence neuronal survival. Together, this body of research suggests that P2X7R may constitute an important therapeutic target for a variety of neurological disorders.

Neuronal subtype-specific transcription factors (TFs) instruct key features of neuronal function and connectivity. Activity-dependent mechanisms also contribute to wiring and circuit assembly, but whether and how they relate to TF-directed neuronal differentiation is poorly investigated. Here we demonstrate that the TF Cux1 controls the formation of the layer II/III corpus callosum (CC) projections through the developmental transcriptional regulation of Kv1 voltage-dependent potassium channels and the resulting postnatal switch to a Kv1-dependent firing mode. Loss of Cux1 function led to a decrease in the expression of Kv1 transcripts, aberrant firing responses, and selective loss of CC contralateral innervation. Firing and innervation were rescued by re-expression of Kv1 or postnatal reactivation of Cux1. Knocking down Kv1 mimicked Cux1-mediated CC axonal loss. These findings reveal that activity-dependent processes are central bona fide components of neuronal TF-differentiation programs and establish the importance of intrinsic firing modes in circuit assembly within the neocortex.

Homeodomain transcription factors play important roles in the specification and differentiation of neuronal subpopulations. In the cerebral cortex, the expression patterns of Cux-1 and Cux-2 in the medial ganglionic eminence (MGE) suggest a role for these transcription factors in the development of interneurons, a heterogeneous neuronal population. In this report, we describe expression of Cux-1 and Cux-2 proteins in Reelin-secreting interneurons of the cortical plate, but not in calretinin or parvalbumin subpopulations. The role of Cux genes in the development of Reelin positive neurons was studied using Cux-1 and Cux-2 knockout mice. These experiments demonstrate that Cux-1À/À; Cux-2À/À double mutation is embryonically lethal. Although this phenotype is highly penetrant, a small proportion of mice develop to birth (P0).Analysis of these animals demonstrate that expression of Reelin is completely absent in layers II-IV of Cux-1À/À; Cux-2À/À double mutant mice, but it is not affected in the cortex of Cux-1À/À or Cux-2À/À single mutants. No Cux-1À/À; Cux-2À/À double-mutant were collected after P0. Since, GABA-ergic populations mature at late postnatal stages, this did not allow us to analyze the expression of subclass specific markers and define the affected interneuron subpopulations. Our analysis of Cux-1À/À; Cux-2À/À double mutant thus demonstrates essential yet redundant roles for Cux-1 and Cux-2 in specifying Reelin expressing cortical interneurons. ' 2008 Wiley Periodicals, Inc. Develop Neurobiol 68: 917-925, 2008

Alzheimer disease is a neurodegenerative disease characterized by the presence of senile plaques composed of amyloid-β (Aβ) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have revealed that extracellular ATP, through its selective receptor P2X7 (P2X7R), is essential to neuroinflammation and neurotoxicity induced by Aβ. P2X7R is upregulated on microglial cells around the senile plaques. This upregulation progressively rises with age and is parallel with an accumulation of senile plaques and also correlates with the synaptic toxicity detected both in animal models reproducing AD and human patients of AD. Furthermore, the late onset of the first AD-associated symptoms suggests that aging associated-changes may be relevant to the disease progression. Thus, microglia motility and its capacity to respond to exogenous ATP stimulus decrease with aging. To evaluate whether the P2X7R age related-changes on microglia cells may be relevant to the AD progression, we generated a new transgenic mouse model crossing an Aβ peptide mouse model, J20 mice and the P2X7R reporter mice P2X7R EGFP. Our results indicate that neuroinflammation induced by Aβ peptide causes changes in the P2X7R distribution pattern, increasing it s expression in microglial cells at advanced and late stages, when microgliosis occurs, but not in the early stages, in the absence of microgliosis. In addition, we found that P2X7R activation promotes microglial cells migration to senile plaques but decreases their phagocytic capacity. Moreover, we found a significant reduction of P2X7R transcription on neuronal cells at the early and advanced stages, but not at the late stages. Since previous studies have reported that either pharmacological inhibition or selective downregulation of P2X7R significantly improve behavioral alterations and reduce the incidence and size of senile plaques in the early and advanced stages of AD, the results presented here provide new evidence, indicating that this therapeutic approach could be also efficient in the late stages of the disease.

Hypomorphic mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme, ALPL in human or Akp2 in mice, cause hypophosphatasia (HPP), an inherited metabolic bone disease also characterized by spontaneous seizures. Initially, these seizures were attributed to the impairment of GABAergic neurotransmission caused by altered vitamin B6 (vit-B6) metabolism. However, clinical cases in human newborns and adults whose convulsions are refractory to pro-GABAergic drugs but controlled by the vit-B6 administration, suggest that other factors are involved. Here, to evaluate whether neurodevelopmental alterations are underlying the seizures associated to HPP, we performed morphological and functional characterization of postnatal homozygous TNAP null mice, a model of HPP. These analyses revealed that TNAP deficient mice present an increased proliferation of neural precursors, an altered neuronal morphology, and an augmented neuronal activity. We found that these alterations were associated with a partial downregulation of the purinergic P2X7 receptor (P2X7R). Even though deficient P2X7R mice present similar neurodevelopmental alterations, they do not develop neonatal seizures. Accordingly, we found that the additional blockage of P2X7R prevent convulsions and extend the lifespan of mice lacking TNAP. In agreement with these findings, we also found that exogenous administration of ATP or TNAP antagonists induced seizures in adult wild-type mice by activating P2X7R. Finally, our results also indicate that the anticonvulsive effects attributed to vit-B6 may be due to its capacity to block P2X7R. Altogether, these findings suggest that the purinergic signalling regulates the neurodevelopmental alteration and the neonatal seizures associated to HPP.

Tissue-nonspecific alkaline phosphatase (TNAP) is one of the four isozymes in humans and mice that have the capacity to hydrolyze phosphate groups from a wide spectrum of physiological substrates. Among these, TNAP degrades substrates implicated in neurotransmission. Transgenic mice lacking TNAP activity display the characteristic skeletal and dental phenotype of infantile hypophosphatasia, as well as spontaneous epileptic seizures and die around 10 days after birth. This physiopathology, linked to the expression pattern of TNAP in the central nervous system (CNS) during embryonic stages, suggests an important role for TNAP in neuronal development and synaptic function, situating it as a good target to be explored for the treatment of neurological diseases. In this review, we will focus mainly on the role that TNAP plays as an ectonucleotidase in CNS regulating the levels of extracellular ATP and consequently purinergic signaling.