Transient Overexposure of Neuregulin 3 during Early Postnatal Development Impacts Selective Behaviors in Adulthood

<div><p>Neuregulin 3 (NRG3), a specific ligand for ErbB4 and a neuronal-enriched neurotrophin is implicated in the genetic predisposition to a broad spectrum of neurodevelopmental, neurocognitive and neuropsychiatric disorders, including Alzheimer's disease, autism and schizophrenia. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, accompanied by increased expression of prefrontal cortical NRG3. Despite our expanding knowledge of genetic involvement of NRG3 in neurological disorders, little is known about the neurodevelopmental mechanisms of risk. Here we exploited the fact that a paralog of NRG3, NRG1, readily penetrates the murine blood brain barrier (BBB). In this study we synthesized the bioactive epidermal growth factor (EGF) domain of NRG3, and using previously validated <i>in-vivo</i> peripheral injection methodologies in neonatal mice, demonstrate that NRG3 successfully crosses the BBB, where it activates its receptor ErbB4 and downstream Akt signaling at levels of bioactivity comparable to NRG1. To determine the impact of NRG3 overexpression during one critical developmental window, C57BL/6 male mice were subcutaneously injected daily with NRG1-EGF, NRG3-EGF or vehicle from postnatal days 2–10. Mice were tested in adulthood using a comprehensive battery of behavioral tasks relevant to neurocognitive and psychiatric disorders. In agreement with previous studies, developmental overexposure to NRG1 induced multiple non-CNS mediated peripheral effects as well as severely disrupting performance of prepulse inhibition of the startle response. In contrast, NRG3 had no effect on any peripheral measures investigated or sensorimotor gating. Specifically, developmental NRG3 overexposure produced an anxiogenic-like phenotype and deficits in social behavior in adulthood. These results provide primary data to support a role for NRG3 in brain development and function, which appears to be distinct from its paralog NRG1. Furthermore we demonstrate how perturbations in NRG3 expression at distinct developmental stages may contribute to the neurological deficits observed in brain disorders such as schizophrenia and autism.</p></div

Effects of neonatal peripheral overexposure to NRG1 and NRG3 on developmental milestones and general health in adulthood.

<p>Following daily injections of Vehicle, 1 mg/kg NRG1 or 3 mg/kg NRG3 mice were assayed for neurodevelopmental milestones, including eye opening and tooth eruption, general health, motoric ability and reflexes in adulthood. N = 9–14/treatment group. Data represent mean ± s.e.m.</p><p>*p<0.05,</p><p>***p<0.001 vs. Vehicle treated mice;</p>#<p>p<0.05.</p>###<p>p<0.001 vs. 3 mg/kg NRG3-EGF treated mice.</p

The EGF domain of NRG3 permeates the neonatal mouse BBB in a dose dependent manner, activating ErbB4-Akt signaling.

<p>Activation levels of pErbB4 (A), and pAktSer473 (B) in hemi-brain lysates of PND2 mice 3 hours following s.c. injection of vehicle (PBS/0.1% BSA), 1–3 mg/kg NRG3-EGF or 1 mg/kg NRG1-EGF. Representative blots of tErbB4, pErbB4, tAKT and pAKT ser473 protein levels are depicted (upper panels). N = 4–5/treatment group, data represents mean± s.e.m ratio of the respective phosphorylated/total protein expression relative to vehicle treatment group. *p<0.05, **p<0.01 compared to vehicle treated group.</p

Overexposure to NRG3 during the neonatal period induces an anxiety-like phenotype in adulthood, but has no impact on locomotor activity or temporal order recency discrimination.

<p>(A) Total distance travelled in 5 minute intervals over a 1 hr exposure to the open field arena did not differ between NRG3-EGF or Vehicle treated mice (n = 12–14/treatment group). (B)Time spent in the center of the open field over a 1 hr period was significantly reduced in mice treated with NRG3-EGF as neonates compared to control treated mice (n = 12–14/treatment group). (C) Representative track plots of exploration in the open field of vehicle (left panel) and NRG3-EGF (right panel) overexposed mice. Inner area designated by white dashed line designates the center arena. (D) Adult mice treated with NRG3-EGF or vehicle during PND 2–10 performed successfully in the temporal order object recognition task of recency discrimination as adults (Discrimination ratio >0, n = 11–14/treatment group). (E) After a 10 minute period of habituation to the open field (indicated by dashed vertical line), mice neonatally overexposed to NRG3-EGF or vehicle were given a single i.p. injection of saline or 3 mg/kg amphetamine. No effect of treatment (NRG3-EGF) was observed on total distance travelled in 5 minute intervals of the subsequent 75 minutes in the context of amphetamine at a dose of 3 mg/kg (n = 6–7/group). Data represents mean ± s.e.m., *p<0.05 compared to vehicle treated mice.</p

Neonatal overexposure to NRG1 but not NRG3 impairs sensorimotor gating in adulthood.

<p>The effect of daily treatment from PND 2–10 with 3 mg/kg NRG3-EGF (A, B) or 1 mg/kg NRG1-EGF (C, D) on startle responses to the presentation of no stimulus or a 120 dB stimulus (A, C) and % prepulse inhibition of the startle response to the presentation of prepulse stimuli at amplitudes of 74, 78, 82, 86 and 90 dB (B, D) in adulthood. Data represents mean ± s.e.m., n = 8–14/treatment group. **p<0.01, main effect of treatment.</p

PKBγ/AKT3 loss-of-function causes learning and memory deficits and deregulation of AKT/mTORC2 signaling: Relevance for schizophrenia

<div><p>Psychiatric genetic studies have identified genome-wide significant loci for schizophrenia. The AKT3/1q44 locus is a principal risk region and gene-network analyses identify AKT3 polymorphisms as a constituent of several neurobiological pathways relevant to psychiatric risk; the neurobiological mechanisms remain unknown. AKT3 shows prenatal enrichment during human neocortical development and recurrent copy number variations involving the 1q43-44 locus are associated with cortical malformations and intellectual disability, implicating an essential role in early brain development. Here, we investigated the role of AKT3 as it relates to aspects of learning and memory and behavioral function, relevant to schizophrenia and cognitive disability, utilizing a novel murine model of Akt3 genetic deficiency. Akt3 heterozygous (Akt3^-/+) or null mice (Akt3^-/-) were assessed in a comprehensive test battery. Brain biochemical studies were conducted to assess the impact of Akt3 deficiency on cortical Akt/mTOR signaling. Akt3^-/+ and Akt3^-/- mice exhibited selective deficits of temporal order discrimination and spatial memory, tasks critically dependent on intact prefrontal-hippocampal circuitry, but showed normal prepulse inhibition, fear conditioned learning, memory for novel objects and social function. Akt3 loss-of-function, reduced brain size and dramatically impaired cortical Akt Ser⁴⁷³ activation in an allele-dose dependent manner. Such changes were observed in the absence of altered Akt1 or Akt2 protein expression. Concomitant reduction of the mTORC2 complex proteins, Rictor and Sin1 identifies a potential mechanism. Our findings provide novel insight into the neurodevelopmental role of Akt3, identify a non-redundant role for Akt3 in the development of prefrontal cortical-mediated cognitive function and show that Akt3 is potentially the dominant regulator of AKT/mTOR signaling in brain.</p></div

Akt3-deficiency does not impact fear learning and extinction, or social behavior.

<p>(a) Freezing behavior prior (baseline), subsequent (post training), and 24 hours later (context 24hr) after the cue-shock pairings. All genotypes increased freezing subsequent to the CS-US pairings and genotypes did not differ during the testing of contextual conditioning (n = 12 WT, 20 Het, 8 KO). (b) Freezing behavior during testing for cued conditioning in the altered context without (CS off) and plus the auditory cue (CS on; n = 12 WT, 20 Het, 8 KO). (c) Fear extinction after repeated exposure to the auditory cue (CS alone) shows no effect of genotype of genotype x day interaction (n = 13 WT, 13 Het, 9 KO) (d) Time spent sniffing the novel mouse during the 10-min test for sociability was greater than time spent sniffing the novel object for all mice (n = 10 WT, 18 Het, 8 KO). (e) Time spent sniffing the novel mouse 2 was greater than time spent sniffing novel mouse 1 (familiar) during the 10-min test for social novelty was greater for all mice (n = 10 WT, 18 Het, 8 KO). ***p≤0.001 main effect of chamber. (a,b,d,e) Data bars represent the mean, with individual data points representing individual subject measures. (c) Data represents mean ± SEM.</p

Impaired temporal order object discrimination memory and spatial location memory in Akt3-deficient mice.

<p>(a) Discrimination ratio displayed by Akt3-deficient mice during the 5 min test phase (sample 3) of the temporal order object recognition task. (** p≤0.01 by posthoc LSD; n = 14 WT, 28 Het, 7 KO). (b) Total time spent exploring the objects presented during the 5-min sample phases 1, 2 and 3. (c) Discrimination ratio for the novel object by Akt3-deficient mice during the novel object test (n = 9 WT, 11 Het, 7 KO). (d) Time spent exploring the two objects during the 10 min acquisition session and the 5 min retention session of the novel object recognition test. (e) Discrimination ratio for the moved object over the same-placed object (**p≤0.01; n = 9 WT, 10 Het, 6 KO). (f) Time spent exploring the two objects during the 10 min acquisition and the 5 min test session of the same object location test. (g) Absence of differences in baseline movement (Nulstim) or Startle responses in the pre-pulse inhibition test in Akt3-deficient mice. (n = 18 WT, 26 Het, 8 KO). (h) Percentage prepulse inhibition of the acoustic startle response displayed by the same mice after the presentation of prepulse (n = 18 WT, 26 Het, 8 KO). Data bars represent the mean, with individual data points representing individual subject measures.</p

Increased locomotor activity but no anxiety-like phenotype in Akt3^-/- mice.

<p>(a) Ambulatory distance in 5 min bins displayed by WT and Akt3-deficient mice over the 60 minutes in the open field arena. (b) Akt3^-/- (KO) mice display a hyperlocomotive phenotype, as defined by a significant increase in total distance traveled per segment. (c) Hyperlocomotion was accounted for by significant increased distance traveled in the border of the open field apparatus. (d) Representative screen shot of a WT and KO mouse performing the open field task. (e) No effect of genotype on time spent in the center of the apparatus was observed. (a) Data represents mean ± SEM. (b, c, e) Data bars represent the mean, with individual data points representing individual subject measures, n = 29 WT, 28 Het, 15 KO. **p≤0.002 compared to WT.</p