Complimentary vertebrate Wac models exhibit phenotypes relevant to DeSanto-Shinawi Syndrome.

Monogenic syndromes are associated with neurodevelopmental changes that result in cognitive impairments, neurobehavioral phenotypes including autism and attention deficit hyperactivity disorder (ADHD), and seizures. Limited studies and resources are available to make meaningful headway into the underlying molecular mechanisms that result in these symptoms. One such example is DeSanto-Shinawi Syndrome (DESSH), a rare disorder caused by pathogenic variants in the WAC gene. Individuals with DESSH syndrome exhibit a recognizable craniofacial gestalt, developmental delay/intellectual disability, neurobehavioral symptoms that include autism, ADHD, behavioral difficulties and seizures. However, no thorough studies from a vertebrate model exist to understand how these changes occur. To overcome this, we developed both murine and zebrafish Wac/wac deletion mutants and studied whether their phenotypes recapitulate those described in individuals with DESSH syndrome. We show that the two Wac models exhibit craniofacial and behavioral changes, reminiscent of abnormalities found in DESSH syndrome. In addition, each model revealed impacts to GABAergic neurons and further studies showed that the mouse mutants are susceptible to seizures, changes in brain volumes that are different between sexes and relevant behaviors. Finally, we uncovered transcriptional impacts of Wac loss of function that will pave the way for future molecular studies into DESSH. These studies begin to uncover some biological underpinnings of DESSH syndrome and elucidate the biology of Wac, with advantages in each model.

Hyperexcitability and translational phenotypes in a preclinical mouse model of SYNGAP1-Related Intellectual Disability.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability, motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicting the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated primary neurons from Syngap1+/- mice displayed increased network firing activity, greater bursts, and shorter inter-burst intervals between peaks by employing high density microelectrode arrays (HD-MEA). Our work bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1 -related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1+/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1+/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1 RI-D, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1 +/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1 +/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1R-ID, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior.

The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.

Parallel functional testing identifies enhancers active in early postnatal mouse brain.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice.

BACKGROUND: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified. METHODS: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior. RESULTS: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits. CONCLUSIONS: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.

Sequential perturbations to mouse corticogenesis following in utero maternal immune activation.

In utero exposure to maternal immune activation (MIA) is an environmental risk factor for neurodevelopmental and neuropsychiatric disorders. Animal models provide an opportunity to identify mechanisms driving neuropathology associated with MIA. We performed time-course transcriptional profiling of mouse cortical development following induced MIA via poly(I:C) injection at E12.5. MIA-driven transcriptional changes were validated via protein analysis, and parallel perturbations to cortical neuroanatomy were identified via imaging. MIA-induced acute upregulation of genes associated with hypoxia, immune signaling, and angiogenesis, by 6 hr following exposure. This acute response was followed by changes in proliferation, neuronal and glial specification, and cortical lamination that emerged at E14.5 and peaked at E17.5. Decreased numbers of proliferative cells in germinal zones and alterations in neuronal and glial populations were identified in the MIA-exposed cortex. Overall, paired transcriptomic and neuroanatomical characterization revealed a sequence of perturbations to corticogenesis driven by mid-gestational MIA.

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

Effects of variable durations of food availability on interval time-place learning with pigeons Columba Livia.

We assessed the effects of variable durations of food availability on an interval time-place learning task. 3 pigeons were exposed to a task in which food could be obtained for responses in one of four feeders according to an RI 25 s during 3 min, after which, food could be obtained on a different feeder according to the same schedule. The correct feeder changed following a fixed sequence that was repeated four times throughout the session. After 50 training sessions, an Open Hopper Test was conducted, after which, the second training condition ensued. This condition was like the first one with the exception that the availability period could be either 1,2,3, or 6 min long. A second test was conducted after 50 sessions of this training. Another group of 3 birds experienced these conditions in the reverse order. Data suggest that birds solved the task via interval timing under the fixed duration condition, and via ordinal timing when faced with variable durations. Birds learned the fixed sequence involved in the task under both conditions. Although the present data agree with previous research exploring variability in TPL tasks, they do not necessarily support previous claims for an asymmetrical role of spatial and temporal information in these tasks.

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome.

BACKGROUND: Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology. RESULTS: Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development. CONCLUSIONS: Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this mouse model. We have noted that a number of the dysregulated genes have known roles in brain development as well as epidermal differentiation and maintenance. Therefore, this study provides clues as to the underlying mechanisms that may be involved in the broader profile of WBS.

The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation.

GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.

Characterization of a Trpc6 Transgenic Mouse Associated with Early Onset FSGS.

RATIONALE: Mutations in Transient Receptor Potential Channel 6 (TRPC6) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations. METHODOLOGY: In order to obtain a time-effective mouse model for Trpc6-associated FSGS we generated a new transgenic mutant Trpc6 mouse model emulating the amino acid change carried by the first pediatric patient of FSGS associated with a TRPC6 mutation: M132T. RESULTS: Mice carrying the orthologous Trpc6 M131T transgene showed early onset proteinuria and early signs of FSGS. When exploring molecular consequences of the overexpression of this mutated form of Trpc6 in podocytes, differences in expression levels of Axin2 and ß-catenin were found in glomeruli from transgenic Trpc6 M131T mice. These data supports the proposed molecular mechanisms related to the activation of calcineurin-NFAT/Wnt signaling, as outcome of the increased calcium influx caused by the mutated form of Trpc6. CONCLUSION: Given that the Trpc6 M131T mouse develops an early onset of FSGS-like phenotypes it represents a promising model for studying the pathogenesis of FSGS caused by TRpC6, facilitating the assessment of new drugs as treatments and allowing further studies to understand underlying molecular pathways involved in the development of the TRPC6 mediated disease.

The role of GTF2IRD1 in the auditory pathology of Williams-Beuren Syndrome.

Williams-Beuren Syndrome (WBS) is a rare genetic condition caused by a hemizygous deletion involving up to 28 genes within chromosome 7q11.23. Among the spectrum of physical and neurological defects in WBS, it is common to find a distinctive response to sound stimuli that includes extreme adverse reactions to loud, or sudden sounds and a fascination with certain sounds that may manifest as strengths in musical ability. However, hearing tests indicate that sensorineural hearing loss (SNHL) is frequently found in WBS patients. The functional and genetic basis of this unusual auditory phenotype is currently unknown. Here, we investigated the potential involvement of GTF2IRD1, a transcription factor encoded by a gene located within the WBS deletion that has been implicated as a contributor to the WBS assorted neurocognitive profile and craniofacial abnormalities. Using Gtf2ird1 knockout mice, we have analysed the expression of the gene in the inner ear and examined hearing capacity by evaluating the auditory brainstem response (ABR) and the distortion product of otoacoustic emissions (DPOAE). Our results show that Gtf2ird1 is expressed in a number of cell types within the cochlea, and Gtf2ird1 null mice showed higher auditory thresholds (hypoacusis) in both ABR and DPOAE hearing assessments. These data indicate that the principal hearing deficit in the mice can be traced to impairments in the amplification process mediated by the outer hair cells and suggests that similar mechanisms may underpin the SNHL experienced by WBS patients.

Comportamiento inteligente y creativo: efectos de distintos tipos de instrucciones / Intelligent and creative behavior: effects of different types of instructions

En este estudio se evaluaron los efectos de distintos tipos de instrucciones en el desempeño en el entrenamiento, pruebas de transferencia y pruebas de comportamiento creativo empleando procedimientos de discriminación condicional. Se distribuyó a 20 estudiantes aleatoriamente en cuatro grupos que recibieron distintos tipos de instrucciones: (a) instrucciones inespecíficas; (b) instrucciones instanciales; (c) instrucciones relacionales parciales, y (d) instrucciones relacionales completas. Los resultados confirman que las instrucciones inespecíficas se correlacionan con desempeños poco efectivos en el entrenamiento, mientras que las instrucciones instanciales propician una mayor efectividad en el entrenamiento, incluso superior que las instrucciones relacionales (parciales y completas), aunque estas se asociaron con mejores desempeños en las pruebas de transferencia y de comportamiento creativo. Se discute que la efectividad del desempeño en que se asienta el ulterior desarrollo del comportamiento inteligente y creativo se adquiere a partir de contactos relacionales con las contingencias de la tarea propiciados por las instrucciones relacionales.

This study assessed the effects of different types of instructions on performance in training, transfer tests and creative behavior testing, using conditional discrimination procedures. Twenty students were randomly assigned to four groups that received different types of instructions: (a) non-specific instructions; (b) instantiated instructions; (c) partial relational instructions, and (d) complete relational instructions. The results showed that non-specific instructions were correlated with ineffective performance in training, while instantiated instructions promoted greater effectiveness in training - even higher than relational instructions (partial or total) - although the latter were associated with the best performance in transfer tests and creative behavior. The article discusses how the effectiveness of performance forms the basis for the subsequent development of intelligent and creative behaviors and is acquired from relational contacts with the contingencies of the task, as fostered by relational instructions.

RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome.

Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

Definition of a critical genetic interval related to kidney abnormalities in the Potocki-Lupski syndrome.

Potocki-Lupski syndrome is a genomic disorder caused by duplication of 17p11.2. It is characterized by failure to thrive, intellectual disability, hypotonia, and behavioral difficulties. Structural renal anomalies have been observed in <10% of affected individuals. We present detailed clinical and molecular data on six patients with Potocki-Lupski syndrome, two of whom had renal abnormalities, and investigate the prevalence of kidney abnormalities in the mouse model for the syndrome. In contrast to affected humans, the mouse model does not demonstrate a renal phenotype. Comparison of the duplicated segment in patients with Potocki-Lupski syndrome and the renal phenotype and the syntenic duplicated region in the mouse model allowed us to suggest a 0.285 Mb critical region, including the FLCN gene that may be important for development of renal abnormalities in patients with this duplication.

Copy number variation and susceptibility to complex traits.

Copy number variations (CNV) within the genome are extremely abundant. In this closeup, Canales and Walz discuss how CNV are associated with normal variation, genomic disorders, genome evolution, adaptive traits and how the use of a novel screen described by Ermakova et al in this issue that is designed to identify human diseaserelevant phenotypes associated with CNV in the mouse can help elucidating susceptibility or predisposition to diseases loci.

Podocyte-specific overexpression of wild type or mutant trpc6 in mice is sufficient to cause glomerular disease.

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.