Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 (SMN2) mRNA Splicing and Expression.

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 (SMN1) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 (SMN2) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3, for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development.

PI3K-Akt Signaling in the Basolateral Amygdala Facilitates Traumatic Stress Enhancements in Fear Memory.

BACKGROUND: A core symptom of posttraumatic stress disorder is persistent fear memory, which can be defined as fear memory that is resistant to updating, inhibition, or extinction. posttraumatic stress disorder emerges after traumatic stress exposure, but neurobiological mechanisms via which traumatic stress leads to persistent fear memory are not well defined. Akt signaling within the amygdala (Amy) is enhanced with traumatic stress, and phosphatidylinositol kinase 3 (PI3K) activation of Akt within the basolateral Amy (BLA) has been implicated as critical to fear memory formation. These findings raise the possibility that traumatic stress enhances PI3K→Akt signaling in the BLA, which leads to persistent fear memory. METHODS: To test this hypothesis, rats were exposed to traumatic stress using the single prolonged stress model, and changes in Akt phosphorylation were assayed in the Amy at 0 and 30 minutes after fear conditioning (FC). In a separate experiment, we inhibited PI3K→Akt signaling in the BLA prior to FC and observed the effect this had on acquisition, expression, and extinction of FC in stressed and control rats. RESULTS: Enhanced Akt phosphorylation in the Amy at both time points was observed in stressed rats, but not in control rats. PI3K→Akt inhibition in the BLA had no effect on freezing in control rats but decreased freezing during extinction training and testing in stressed rats. CONCLUSION: These findings suggest that PI3K→Akt signaling in the BLA could be a mechanism via which traumatic stress leads to fear memory that is resistant to extinction.

Factor Analysis of the Childhood Autism Rating Scale in a Sample of Two Year Olds with an Autism Spectrum Disorder.

The Childhood Autism Rating Scale (CARS), (Schopler et al. in J Autism Dev Disord 10(1):91-103, 1980) is a 15-item observation-based rating scale that yields a total score reflective of autism symptom severity. This study investigated the factor structure of the CARS in a sample of 2-year-old children with DSM-IV-TR (American Psychiatric Association in Diagnostic and statistical manual of mental disorders, 4th edn. American Psychiatric Publishing, Washington, 2000) diagnoses of AD or PDD-NOS. Following a preliminary internal cross-validation, principal axis factor analysis was completed (N = 282). The results indicate a three-factor solution: Social Communication, Stereotyped Behaviors and Sensory Sensitivities, and Emotional Reactivity. The factors are meaningful, with the first two reflective of DSM-5 symptom domains. This study supports the continued relevance of the CARS in ASD assessment, and extends its utility in 2-year-old children.

Characterizing changes in glucocorticoid receptor internalization in the fear circuit in an animal model of post traumatic stress disorder.

Glucocorticoid receptors (GRs) shuttle from the cytoplasm (cy) to the nucleus (nu) when bound with glucocorticoids (i.e. GR internalization) and alter transcriptional activity. GR activation within the fear circuit has been implicated in fear memory and post traumatic stress disorder (PTSD). However, no study to date has characterized GR internalization within the fear circuit during fear memory formation or examined how traumatic stress impacts this process. To address this, we assayed cy and nu GR levels at baseline and after auditory fear conditioning (FC) in the single prolonged stress (SPS) model of PTSD. Cy and nu GRs within the medial prefrontal cortex (mPFC), dorsal hippocampus (dHipp), ventral hippocampus (vHipp), and amygdala (AMY) were assayed using western blot. The distribution of GR in the cy and nu (at baseline and after FC) was varied across individual nodes of the fear circuit. At baseline, SPS enhanced cyGRs in the dHipp, but decreased cyGRs in the AMY. FC only enhanced GR internalization in the AMY and this effect was attenuated by SPS exposure. SPS also decreased cyGRs in the dHipp after FC. The results of this study suggests that GR internalization is varied across the fear circuit, which in turn suggests GR activation is selectively regulated within individual nodes of the fear circuit. The findings also suggest that changes in GR dynamics in the dHipp and AMY modulate the enhancing effect SPS has on fear memory persistence.

Early Characteristics of Children with ASD Who Demonstrate Optimal Progress Between Age Two and Four.

Although for many children, Autism Spectrum Disorder (ASD) is a lifelong disability, a subset of children with ASD lose their diagnosis and show typical cognitive and adaptive abilities. The ages at which this transition can occur is not known, but it sometimes occurs quite early. Participants in the current study were 207 children with an ASD at age two who were reevaluated at age four. Eighty-three percent retained an ASD diagnosis at reevaluation and 9 % showed "optimal progress": clear ASD at age two but not at age four, and average cognition, language, communication and social skills at age four. Early child-level factors predicted optimal progress: diagnosis of PDD-NOS, fewer repetitive behaviors, less severe symptomatology and stronger adaptive skills.

The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts.

BACKGROUND: Most previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies. RESULTS: From reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection. CONCLUSIONS: Chromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.