Fragile X syndrome (FXS) is the most frequent form of heritable intellectual disability and autism. Fragile X (Fmr1-KO) mice exhibit aberrant dendritic spine structure, synaptic plasticity, and cognition. Autophagy is a catabolic process of programmed degradation and recycling of proteins and cellular components via the lysosomal pathway. However, a role for autophagy in the pathophysiology of FXS is, as yet, unclear. Here we show that autophagic flux, a functional readout of autophagy, and biochemical markers of autophagy are down-regulated in hippocampal neurons of fragile X mice. We further show that enhanced activity of mammalian target of rapamycin complex 1 (mTORC1) and translocation of Raptor, a defining component of mTORC1, to the lysosome are causally related to reduced autophagy. Activation of autophagy by delivery of shRNA to Raptor directly into the CA1 of living mice via the lentivirus expression system largely corrects aberrant spine structure, synaptic plasticity, and cognition in fragile X mice. Postsynaptic density protein (PSD-95) and activity-regulated cytoskeletal-associated protein (Arc/Arg3.1), proteins implicated in spine structure and synaptic plasticity, respectively, are elevated in neurons lacking fragile X mental retardation protein. Activation of autophagy corrects PSD-95 and Arc abundance, identifying a potential mechanism by which impaired autophagy is causally related to the fragile X phenotype and revealing a previously unappreciated role for autophagy in the synaptic and cognitive deficits associated with fragile X syndrome.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disabilities and a leading cause of autism. FXS is caused by a trinucleotide expansion in the gene FMR1 on the X chromosome. The neuroanatomical hallmark of FXS is an overabundance of immature dendritic spines, a factor thought to underlie synaptic dysfunction and impaired cognition. We showed that aberrantly increased activity of the Rho GTPase Rac1 inhibited the actin-depolymerizing factor cofilin, a major determinant of dendritic spine structure, and caused disease- associated spine abnormalities in the somatosensory cortex of FXS model mice. Increased cofilin phosphorylation and actin polymerization coincided with abnormal dendritic spines and impaired synaptic maturation. Viral delivery of a constitutively active cofilin mutant (cofilinS3A) into the somatosensory cortex of Fmr1-deficient mice rescued the immature dendritic spine phenotype and increased spine density. Inhibition of the Rac1 effector PAK1 with a small-molecule inhibitor rescued cofilin signaling in FXS mice, indicating a causal relationship between PAK1 and cofilin signaling. PAK1 inhibition rescued synaptic signaling (specifically the synaptic ratio of NMDA/AMPA in layer V pyramidal neurons) and improved sensory processing in FXS mice. These findings suggest a causal relationship between increased Rac1-cofilin signaling, synaptic defects, and impaired sensory processing in FXS and uncover a previously unappreciated role for impaired Rac1-cofilin signaling in the aberrant spine morphology and spine density associated with FXS.

Background Prenatal ethanol (EtOH) and prenatal stress have both been independently shown to induce learning deficits and anxiety behavior in adult offspring. However, the interactive effects of these two developmental teratogens on behavioral outcomes have not been systematically evaluated. Methods We combined an established moderate prenatal EtOH consumption paradigm where Long-Evans rat dams voluntarily consume either a 0% or 5% EtOH solution in 0.066% saccharin water (resulting in a mean peak maternal serum EtOH concentration of 84 mg/dL) with a novel prenatal stress paradigm. Pregnant rats were exposed to 3% 2,3,5-trimethyl-3-thiazoline (TMT) for 20 minutes a day on Gestational Days 13, 15, 17, and 19. Adult female offspring were evaluated for anxiety-like behavior using an elevated plus maze and hippocampal-sensitive learning using a two-trial trace conditioning task. Results TMT exposure produced a three-fold increase in maternal serum corticosterone compared to non-exposed, unhandled controls. Neither prenatal exposure paradigm, either alone or in combination, altered maternal weight gain, ethanol consumption, maternal care of litters, litter size, pup birth weight, or pup weight gain up to weaning. Offspring exposed to prenatal stress displayed significant increases in anxiety-like behavior in the elevated plus maze in terms of open arm entries and time spent on the open arms, with no significant effect of prenatal EtOH exposure and no interaction of the two prenatal exposures. Performance in a two-trial trace conditioning task revealed a significant effect of prenatal ethanol exposure on freezing behavior on the testing day, with no significant effect of prenatal stress exposure and no interaction of the two prenatal exposures. Conclusions While each prenatal exposure independently produced different behavioral outcomes, the results indicate that there is no significant interaction of prenatal ethanol and prenatal stress exposures on learning or anxiety at the exposure levels employed in this dual exposure paradigm. Subsequent studies will examine whether similar outcomes occur in male offspring and whether other measures of anxiety or learning are differentially impacted by these prenatal exposure paradigms.

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Jegou et al. (2012) have reported prenatal alcohol exposure (PAE)-induced reductions of angiogenesis-related proteins in mouse placenta. These effects were associated with striking alterations in microvascular development in neonatal cerebral cortex. Here, we employed a rat model of moderate PAE to search for additional proteins whose placental and fetal cortical expression is altered by PAE, along with a subsequent examination of fetal cerebral cortical alterations associated with altered protein expression. Long-Evans rat dams voluntarily consumed either a 0 or 5% ethanol solution 4 h each day throughout gestation. Daily ethanol consumption, which resulted in a mean peak maternal serum ethanol concentration of 60.8 mg/dL, did not affect maternal weight gain, litter size, or placental or fetal body weight. On gestational day 20, rat placental: fetal units were removed by Caesarian section. Placental protein expression, analyzed by 2D-PAGEtandem mass spectroscopy, identified a total of 1,117 protein spots, 20 of which were significantly altered by PAE. To date, 14 of these PAE-altered proteins have been identified. Western blotting confirmed the alterations of two of these placental proteins, namely, annexin-A4 (ANX-A4) and cerebral cavernous malformation protein 3 (CCM-3). Specifically, PAE elevated ANX-A4 and decreased CCM-3 in placenta. Subsequently, these two proteins were measured in fetal cerebral cortex, along with radiohistochemical studies of VEGF binding and histofluorescence studies of microvascular density in fetal cerebral cortex. PAE elevated ANX-A4 and decreased CCM-3 in fetal cerebral cortex, in a pattern similar to the alterations observed in placenta. Further, both VEGF receptor binding and microvascular density and orientation, measures that are sensitive