Evidence for activation of the unfolded protein response in collagen IV nephropathies.

Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.

Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy.

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.

Sox1 is required for the specification of a novel p2-derived interneuron subtype in the mouse ventral spinal cord.

During mouse development, the ventral spinal cord becomes organized into five progenitor domains that express different combinations of transcription factors and generate different subsets of neurons and glia. One of these domains, known as the p2 domain, generates two subtypes of interneurons, V2a and V2b. Here we have used genetic fate mapping and loss-of-function analysis to show that the transcription factor Sox1 is expressed in, and is required for, a third type of p2-derived interneuron, which we named V2c. These are close relatives of V2b interneurons, and, in the absence of Sox1, they switch to the V2b fate. In addition, we show that late-born V2a and V2b interneurons are heterogeneous, and subsets of these cells express the transcription factor Pax6. Our data demonstrate that interneuron diversification in the p2 domain is more complex than previously thought and directly implicate Sox1 in this process.

Spatially distinct functions of PAX6 and NKX2.2 during gliogenesis in the ventral spinal cord.

During ventral spinal cord (vSC) development, the p3 and pMN progenitor domain boundary is thought to be maintained by cross-repressive interactions between NKX2.2 and PAX6. Using loss-of-function analysis during the neuron-glial fate switch we show that the identity of the p3 domain is not maintained by the repressive function of NKX2.2 on Pax6 expression, even in the absence of NKX2.9. We further show that NKX2.2 is necessary to induce the expression of Slit1 and Sulfatase 1 (Sulf1) in the vSC in a PAX6-independent manner. Conversely, we show that PAX6 regulates Sulf1/Slit1 expression in the vSC in an NKX2.2/NKX6.1-independent manner. Consequently, deregulation of Sulf1 expression in Pax6-mutant embryos has stage-specific implications on neural patterning, associated with enhancement of Sonic Hedgehog activity. On the other hand, deregulation of Slit1 expression in gliogenic neural progenitors leads to changes in Astrocyte subtype identity. These data provide important insights into specific functions of PAX6 and NKX2.2 during glial cell specification that have so far remained largely unexplored.

Generation of an ABCG2(GFPn-puro) transgenic line--a tool to study ABCG2 expression in mice.

The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch.

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Nu/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.

Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain.

Studies proposed a model for embryonic neurogenesis where the expression levels of the SOXB2 and SOXB1 factors regulate the differentiation status of the neural stem cells. However, the precise role of the SOXB2 genes remains controversial. Therefore, this study aims to investigate the effects of individual deletions of the SOX21 and SOX14 genes during the development of the dorsal midbrain. We show that SOX21 and SOX14 function distinctly during the commitment of the GABAergic lineage. More explicitly, deletion of SOX21 reduced the expression of the GABAergic precursor marker GATA3 and BHLHB5 while the expression of GAD6, which marks GABAergic terminal differentiation, was not affected. In contrast deletion of SOX14 alone was sufficient to inhibit terminal differentiation of the dorsal midbrain GABAergic neurons. Furthermore, we demonstrate through gain-of-function experiments, that despite the homology of SOX21 and SOX14, they have unique gene targets and cannot compensate for the loss of each other. Taken together, these data do not support a pan-neurogenic function for SOXB2 genes in the dorsal midbrain, but instead they influence, sequentially, the specification of GABAergic neurons.

In vivo monitoring of the inflammatory response in a stented mouse aorta model.

The popularity of vascular stents continues to increase for a variety of applications, including coronary, lower limb, renal, carotid, and neurovascular disorders. However, their clinical effectiveness is hindered by numerous postdeployment complications, which may stimulate inflammatory and fibrotic reactions. The purpose of this study was to evaluate the vessel inflammatory response via in vivo imaging in a mouse stent implantation model. Corroded and noncorroded self-expanding miniature nitinol stents were implanted in mice abdominal aortas, and novel in vivo imaging techniques were used to assess trafficking and accumulation of fluorescent donor monocytes as well as cellular proliferation at the implantation site. Monocytes were quantitatively tracked in vivo and found to rapidly clear from circulation within hours after injection. Differences were found between the test groups with respect to the numbers of recruited monocytes and the intensity of the resulting fluorescent signal. Image analysis also revealed a subtle increase in matrix metalloproteinase activity in corroded compared with the normal stented aortas. In conclusion, this study has been successful in developing a murine stent inflammation model and applying novel in vivo imaging tools and methods to monitor the complex biological processes of the host vascular wall response.

Pax6 is expressed in subsets of V0 and V2 interneurons in the ventral spinal cord in mice.

The embryonic spinal cord in mice is organized into eleven progenitor domains. Cells in each domain first produce neurons and then switch to specifying glia. Five of these domains known as p3, pMN, p2, p1 and p0 are located in the ventral spinal cord and each expresses a unique code of transcription factors (TFs) that define the molecular profile of progenitor cells. This code is largely responsible for determining the subtype specification of neurons generated from each domain. Pax6 codes for a homedomain-containing TF that plays a central role in defining the molecular boundaries between the two ventral-most domains, p3 and pMN. Using fate mapping and gene expression studies we show that PAX6, in addition to each patterning function, is expressed in a group of late born interneurons that derive from the p2 and p0 domains. The p2-derived neurons represent a subset of late born V2b interneurons and their specification depends on Notch signaling. The V0 neurons represent V0v ventral neurons expressing Pax2. Our data demonstrate that interneuron diversity in the ventral spinal cord is more complex than originally appreciated and point to the existence of additional mechanisms that determine interneuron diversity, particularly in the p2 domain.