Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Assemblies of Perisomatic GABAergic Neurons in the Developing Barrel Cortex.

The developmental journey of cortical interneurons encounters several activity-dependent milestones. During the early postnatal period in developing mice, GABAergic neurons are transient preferential recipients of thalamic inputs and undergo activity-dependent migration arrest, wiring, and programmed cell-death. Despite their importance for the emergence of sensory experience and the role of activity in their integration into cortical networks, the collective dynamics of GABAergic neurons during that neonatal period remain unknown. Here, we study coordinated activity in GABAergic cells of the mouse barrel cortex using in vivo calcium imaging. We uncover a transient structure in GABAergic population dynamics that disappears in a sensory-dependent process. Its building blocks are anatomically clustered GABAergic assemblies mostly composed by prospective parvalbumin-expressing cells. These progressively widen their territories until forming a uniform perisomatic GABAergic network. Such transient patterning of GABAergic activity is a functional scaffold that links the cortex to the external world prior to active exploration. VIDEO ABSTRACT.

Magnesium reduces carotid intima-media thickness in a mouse model of pseudoxanthoma elasticum: a novel treatment biomarker.

Pseudoxanthoma elasticum (PXE), which demonstrates progressive build-up of calcium phosphate and proteoglycan deposits in skin, eye, and arteries, has been associated with myocardial infarctions, stroke, and blindness. In a mouse model of PXE, a magnesium-enriched diet prevents mineralization of the vibrissae capsule, an early biomarker for PXE. However, biomarkers for therapeutic responses in PXE have not been identified in humans. Because PXE patients have an increased carotid intima-media thickness (CIMT), a risk factor for cardiovascular disease and stroke, we analyzed the feasibility of CIMT as a treatment endpoint before and after magnesium supplementation in a mouse model of PXE (Abcc6(-/-) ). CIMT was measured in 1-year-old Abcc6(-/-) and Abcc6(+/+) mice fed either standard rodent diet with or without magnesium oxide supplementation for 2 months. Baseline CIMT in Abcc6(-/-) versus Abcc6(+/+) mice was increased (p value = 0.009), whereas CIMT in magnesium-treated versus untreated Abcc6(-/-) mice was reduced (p value = 0.024). CIMT is a novel treatment endpoint in this mouse model and may serve as a predictive biomarker of therapeutic response in PXE patients. In that context, magnesium oxide significantly reduced CIMT in PXE mice, and may be useful for disease prevention in PXE patients.

Physical rehabilitation of paralysed facial muscles: functional and morphological correlates.

Using a combined morphofunctional approach, we recently found that polyinnervation of the neuromuscular junction (NMJ) is the critical factor for recovery of function after transection and suture of the facial nerve. Since polyinnervation is activity-dependent and can be manipulated, we tried to design a clinically feasible therapy by electrical stimulation or by soft tissue massage. First, electrical stimulation was applied to the transected facial nerve or to paralyzed facial muscles. Both procedures did not improve vibrissal motor performance (video-based motion analysis of whisking), failed to diminish polyinnervation, and even reduced the number of innervated NMJ to one-fifth of normal values. In contrast, gentle stroking of the paralyzed vibrissal muscles by hand resulted in full recovery of whisking. Manual stimulation depended on the intact sensory supply of the denervated muscle targets and was also effective after hypoglossal-facial anastomosis, after interpositional nerve grafting, when applied to the orbicularis oculi muscle and after transection and suture of the hypoglossal nerve. From these results, we conclude that manual stimulation is a noninvasive procedure with immediate potential for clinical rehabilitation following facial nerve reconstruction.

Diffuse traumatic brain injury initially attenuates and later expands activation of the rat somatosensory whisker circuit concomitant with neuroplastic responses.

Traumatic brain injury can initiate an array of chronic neurological deficits, effecting executive function, language and sensorimotor integration. Mechanical forces produce the diffuse pathology that disrupts neural circuit activation across vulnerable brain regions. The present manuscript explores the hypothesis that the extent of functional activation of brain-injured circuits is a consequence of initial disruption and consequent reorganization. In the rat, enduring sensory sensitivity to whisker stimulation directs regional analysis to the whisker barrel circuit. Adult, male rats were subjected to midline fluid percussion brain or sham injury and evaluated between 1day and 42days post-injury. Whisker somatosensory regions of the cortex and thalamus maintained cellular composition as visualized by Nissl stain. Within the first week post-injury, quantitatively less cFos activation was elicited by whisker stimulation, potentially due to axotomy within and surrounding the whisker circuit as visualized by amyloid precursor protein immunohistochemistry. Over six weeks post-injury, cFos activation after whisker stimulation showed a significant linear correlation with time in the cortex (r(2)=0.545; p=0.015), non-significant correlation in the thalamus (r(2)=0.326) and U-shaped correlation in the dentate gyrus (r(2)=0.831), all eventually exceeding sham levels. Ongoing neuroplastic responses in the cortex are evidenced by accumulating growth associated protein and synaptophysin gene expression. In the thalamus, the delayed restoration of plasticity markers may explain the broad distribution of neuronal activation extending into the striatum and hippocampus with whisker stimulation. The sprouting of diffuse-injured circuits into diffuse-injured tissue likely establishes maladaptive circuits responsible for behavioral morbidity. Therapeutic interventions to promote adaptive circuit restructuring may mitigate post-traumatic morbidity.

Overexpression of fetuin-a counteracts ectopic mineralization in a mouse model of pseudoxanthoma elasticum (abcc6(-/-)).

The pathologic hallmark of pseudoxanthoma elasticum (PXE) is ectopic mineralization of soft connective tissues. Recent studies have suggested that PXE is a metabolic disease, and perturbations in a number of circulatory factors have been postulated. One of them is fetuin-A, a 60-kDa glycoprotein synthesized in the liver and secreted into blood. Observations in targeted mutant mice (Ahsg(-/-)) and in cell culture model systems have shown that fetuin-A is a powerful anti-mineralization factor in circulation, and the serum levels of fetuin-A in patients with PXE as well as in a mouse model of PXE (Abcc6(-/-)) have been shown to be reduced by up to 30%. In this study, we tested the hypothesis that overexpression of fetuin-A in Abcc6(-/-) mice counteracts the ectopic mineralization. Delivery of an expression construct containing full-length mouse fetuin-A complementary DNA (cDNA), linked to a His-tag, to the liver of these mice resulted in elevated serum levels of this protein. As a consequence, soft tissue mineralization, which is a characteristic of Abcc6(-/-) mice, was reduced by approximately 70% at 12 weeks of age, but the effect was transient when examined 4 weeks later. The results suggest that normalization of serum fetuin-A, either through gene therapy approaches or by direct protein delivery to the circulation, may offer strategies for treating PXE and perhaps other heritable disorders of soft tissue mineralization.

A cell-based system for screening hair growth-promoting agents.

Androgen-inducible transforming growth factor beta (TGF-beta1) derived from dermal papilla cells (DPCs) is a catagen inducer that mediates hair growth suppression in androgenetic alopecia (AGA). In this study, a cell-based assay system was developed to monitor TGF-beta1 promoter activity and then used to evaluate the effects of activated TGF-beta1 promoter in human epidermal keratinocytes (HaCaT). To accomplish this, a pMetLuc-TGF-beta1 promoter plasmid that expresses the luciferase reporter gene in response to TGF-beta1 promoter activity was constructed. Treatment of HaCaT with dihydrotestosterone, which is known to be a primary factor of AGA, resulted in a concentration-dependent increase in TGF-beta1 promoter activity. However, treatment of HaCaT with the TGF-beta1 inhibitor, curcumin, resulted in a concentration-dependant decrease in TGF-beta1 expression. Subsequent use of this assay system to screen TGF-beta1 revealed that HaCaT that were treated with apigenin showed decreased levels of TGF-beta1 expression. In addition, treatment with apigenin also significantly increased the proliferation of both SV40T-DPCs (human DPCs) and HaCaT cells. Furthermore, apigenin stimulated the elongation of hair follicles in a rat vibrissa hair follicle organ culture. Taken together, these findings suggest that apigenin, which is known to have antioxidant, anti-inflammatory, and anti-tumor properties, stimulates hair growth through downregulation of the TGF-beta1 gene. In addition, these results suggest that this assay system could be used to quantitatively measure TGF-beta1 promoter activity in HaCaT, thereby facilitating the screening of agents promoting hair growth.

Magnesium carbonate-containing phosphate binder prevents connective tissue mineralization in Abcc6(-/-) mice-potential for treatment of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues primarily in the skin, eyes, and the cardiovascular system. PXE is caused by mutations in the ABCC6 gene. While PXE is associated with considerable morbidity and mortality, there is currently no effective or specific treatment. In this study, we tested oral phosphate binders for treatment of a mouse model of PXE which we have developed by targeted ablation of the corresponding mouse gene (Abcc6(-/-)). This "knock-out" (KO) mouse model recapitulates features of PXE and demonstrates mineralization of a number of tissues, including the connective tissue capsule surrounding vibrissae in the muzzle skin which serves as an early biomarker of the mineralization process. Treatment of these mice with a magnesium carbonate-enriched diet (magnesium concentration being 5-fold higher than in the control diet) completely prevented mineralization of the vibrissae up to 6 months of age, as demonstrated by computerized morphometric analysis of histopathology as well as by calcium and phosphate chemical assays. The magnesium carbonate-enriched diet also prevented the progression of mineralization when the mice were placed on that experimental diet at 3 months of age and followed up to 6 months of age. Treatment with magnesium carbonate was associated with a slight increase in the serum concentration of magnesium, with no effect on serum calcium and phosphorus levels. In contrast, concentration of calcium in the urine was increased over 10-fold while the concentration of phosphorus was markedly decreased, being essentially undetectable after long-term (> 4 month) treatment. No significant changes were noted in the serum parathyroid hormone levels. Computerized axial tomography scan of bones in mice placed on magnesium carbonate-enriched diet showed no differences in the bone density compared to mice on the control diet, and chemical assays showed a small increase in the calcium and phosphate content of the femurs by chemical assay, in comparison to mice on control diet. Similar experiments with another experimental diet supplemented with lanthanum carbonate did not interfere with the mineralization process in Abcc6(-/-) mice. These results suggest that magnesium carbonate may offer a potential treatment modality for PXE, a currently intractable disease, as well as for other conditions characterized by ectopic mineralization of connective tissues.

Ectopic mineralization of connective tissue in Abcc6-/- mice: effects of dietary modifications and a phosphate binder--a preliminary study.

Pseudoxanthoma elasticum (PXE), a heritable multisystem disorder, is caused by mutations in the ABCC6 gene. We have developed a murine model for PXE by targeted inactivation of the corresponding mouse gene. A feature of this mouse model is ectopic mineralization of connective tissue capsule surrounding the bulb of vibrissae. This study was designed to investigate the effect of dietary sevelamer hydrochloride (Renagel), a phosphate binder, and specific mineral modifications on ectopic mineralization of connective tissue in Abcc6-/- mice. Three groups were fed a specific diet: (i) a standard rodent diet, (ii) a standard rodent diet supplemented with sevelamer hydrochloride, and (iii) a custom experimental diet with specific mineral modifications (high phosphorus, low calcium and low magnesium). The degree of mineralization was determined in hematoxylin-eosin-stained sections using computerized morphometric analysis and by chemical assays to measure the calcium and phosphorus content of the vibrissae. The results indicated increased mineralization in the Abcc6-/- mice fed a standard diet or a diet with mineral modifications as compared with control mice fed a standard diet. However, feeding Abcc6-/- mice with diet supplemented with sevelamer hydrochloride did not improve mineralization, in comparison to mice fed with normal diet. Collectively, these results suggest that the mineralization process in PXE may be exacerbated by changes in mineral intake. The role of dietary minerals, and phosphorus in particular, as well as that of phosphate binders, in ectopic mineralization of PXE, merits further investigation.