p75NTR, but not proNGF, is upregulated following status epilepticus in mice.

ProNGF and p75(NTR) are upregulated and induce cell death following status epilepticus (SE) in rats. However, less is known about the proneurotrophin response to SE in mice, a more genetically tractable species where mechanisms can be more readily dissected. We evaluated the temporal- and cell-specific induction of the proneurotrophins and their receptors, including p75(NTR), sortilin, and sorCS2, following mild SE induced with kainic acid (KA) or severe SE induced by pilocarpine. We found that mature NGF, p75(NTR), and proBDNF were upregulated following SE, while proNGF was not altered, indicating potential mechanistic differences between rats and mice. ProBDNF was localized to mossy fibers and microglia following SE. p75(NTR) was transiently induced primarily in axons and axon terminals following SE, as well as in neuron and astrocyte cell bodies. ProBDNF and p75(NTR) increased independently of cell death and their localization was different depending on the severity of SE. We also examined the expression of proneurotrophin co-receptors, sortilin and sorCS2. Following severe SE, sorCS2, but not sortilin, was elevated in neurons and astrocytes. These data indicate that important differences exist between rat and mouse in the proneurotrophin response following SE. Moreover, the proBDNF and p75(NTR) increase after seizures in the absence of significant cell death suggests that proneurotrophin signaling may play other roles following SE.

Mir-592 regulates the induction and cell death-promoting activity of p75NTR in neuronal ischemic injury.

The neurotrophin receptor p75(NTR) has been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75(NTR) expression after injury. Here, we show that after focal cerebral ischemia in vivo or oxygen-glucose deprivation in organotypic hippocampal slices or neurons, p75(NTR) is rapidly induced. A concomitant induction of proNGF, a ligand for p75(NTR), is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen-glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75(NTR) or proNGF and in p75(NTR) and ngf haploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted in p75(NTR)-/- mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75(NTR). We demonstrate that induction of p75(NTR) after ischemic injury is independent of transcription but requires active translation. Basal levels of p75(NTR) in neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75(NTR) levels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75(NTR) levels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75(NTR) and attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75(NTR) expression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.

ProNGF, a cytokine induced after myocardial infarction in humans, targets pericytes to promote microvascular damage and activation.

Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro-nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75(NTR) in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75(NTR) by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75(NTR) in proNGF-expressing mice rescues the phenotype, confirming the importance of p75(NTR)-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75(NTR) limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.

Divergent stress-induced neuroendocrine and behavioral responses prior to puberty.

Following an acute stressor, pre-adolescent rats exhibit a protracted hormonal response compared to adults, while after repeated exposure to the same stressor (i.e., homotypic stress) prepubertal males fail to habituate like adults. Though the neurobehavioral implications of these changes are unknown, studying pubertal shifts in stress reactivity may help elucidate the mechanisms that underlie the increase in stress-related psychological and physiological disorders often observed during adolescence. Here, we investigated hormonal, behavioral, and neural responses of prepubertal (30d) and adult (77d) male rats before, during, or after acute stress (restraint), homotypic stress (repeated restraint) or heterotypic stress (repeated cold exposure followed by restraint). We found that prepubertal males exhibit prolonged corticosterone responses following acute and heterotypic stress, and higher adrenocorticotropic hormone and corticosterone responses after homotypic stress, compared to adults. Despite these significant age-dependent changes in hormonal responsiveness, we found that struggling behavior during restraint was similar at both ages, such that both prepubertal and adult animals exposed to homotypic stress struggled less than animals exposed to either acute or heterotypic stress. Across these different stress paradigms, we found greater neural activation, as indexed by FOS immunostaining, in the prepubertal compared to adult paraventricular nucleus of the hypothalamus, a nucleus integral for initiating the hormonal stress response. Interestingly, however, we did not find any influence of pubertal development on stress-induced activation of the posterior paraventricular thalamic nucleus, a brain region involved in experience-dependent changes in stress reactivity. Collectively, our data indicate that prepubertal and adult males display divergent hormonal, behavioral, and neural responses following a variety of stressful experiences, as well as a distinct dissociation between hormonal and behavioral reactivity in prepubertal males under homotypic conditions.

DNA pol λ's extraordinary ability to stabilize misaligned DNA.

DNA polymerases have the venerable task of maintaining genome stability during DNA replication and repair. Errors, nonetheless, occur with error propensities that are polymerase specific. For example, DNA polymerase λ (pol λ) generates single-base deletions through template-strand slippage within short repetitive DNA regions much more readily than does the closely related polymerase ß (pol ß). Here we present in silico evidence to help interpret pol λ's greater tendency for deletion errors than pol ß by its more favorable protein/DNA electrostatic interactions immediately around the extrahelical nucleotide on the template strand. Our molecular dynamics and free energy analyses suggest that pol λ provides greater stabilization to misaligned DNA than aligned DNA. Our study of several pol λ mutants of Lys544 (Ala, Phe, Glu) probes the interactions between the extrahelical nucleotide and the adjacent Lys544 to show that the charge of the 544 residue controls stabilization of the DNA misalignment. In addition, we identify other thumb residues (Arg538, Lys521, Arg517, and Arg514) that play coordinating roles in stabilizing pol λ's interactions with misaligned DNA. Interestingly, their aggregate stabilization effect is more important than that of any one component residue, in contrast to aligned DNA systems, as we determined from mutations of these key residues and energetic analyses. No such comparable network of stabilizing misaligned DNA exists in pol ß. Evolutionary needs for DNA repair on substrates with minimal base-pairing, such as those encountered by pol λ in the non-homologous end-joining pathway, may have been solved by a greater tolerance to deletion errors. Other base-flipping proteins share similar binding properties and motions for extrahelical nucleotides.

The effects of stress on play and home cage behaviors in adolescent male rats.

One prominent feature of adolescence is the high frequency of social behaviors, such as play. Engaging in these behaviors appears necessary for proper socio-emotional development as social isolation during adolescence typically leads to behavioral dysfunctions in adulthood. The present experiments examined the effects of stress on social and nonsocial behaviors in group housed adolescent male rats. We found that acute restraint stress led to a complete inhibition of play (e.g., nape contacts and pins) and reduced social investigations in pre- (28 days), mid- (35 days), and late-adolescent (42 and 49 days) animals. A follow-up study, however, found that restraint-induced suppression of play and social investigations was transient such that experimental animals engage in these behaviors at levels similar to those of controls 1 hr after termination of the stressor. We also found that exposure to repeated restraint stress throughout adolescence led to a decrease in social investigations, while leaving play largely unaffected. Interestingly, in all of the experiments, nonsocial behaviors (e.g., eating, drinking, grooming) were unaffected by restraint, suggesting these effects of stress are specific to social behaviors. Together, these data indicate that both acute and repeated stress significantly affect social behaviors during adolescence.