Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3.

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

Haploinsufficiency in peptidylglycine alpha-amidating monooxygenase leads to altered synaptic transmission in the amygdala and impaired emotional responses.

The mammalian amygdala expresses various neuropeptides whose signaling has been implicated in emotionality. Many neuropeptides require amidation for full activation by peptidylglycine α-amidating monooxygenase (PAM), a transmembrane vesicular cuproenzyme and regulator of the secretory pathway. Mice heterozygous for the Pam gene (PAM(+/-)) exhibit physiological and behavioral abnormalities related to specific peptidergic pathways. In the present study, we evaluated emotionality and examined molecular and cellular responses that characterize neurophysiological differences in the PAM(+/-) amygdala. PAM(+/-) mice presented with anxiety-like behaviors in the zero maze that were alleviated by diazepam. PAM(+/-) animals were deficient in short- and long-term contextual and cued fear conditioning and required higher shock intensities to establish fear-potentiated startle than their wild-type littermates. Immunohistochemical analysis of the amygdala revealed PAM expression in pyramidal neurons and local interneurons that synthesize GABA. We performed whole-cell recordings of pyramidal neurons in the PAM(+/-) amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM(+/-) lateral nucleus were deficient in long-term potentiation. This deficit was apparent in the absence and presence of the GABA(A) receptor antagonist picrotoxin and was abolished when both GABA(A) and GABA(B) receptors were blocked. Both evoked and spontaneous excitatory signals were enhanced in the PAM(+/-) lateral nucleus. Phasic GABAergic signaling was also augmented in the PAM(+/-) amygdala, and this difference comprised activity-independent and -dependent components. These physiological findings represent perturbations in the PAM(+/-) amygdala that may underlie the aberrant emotional responses in the intact animal.

Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

Peptidylgycine α-amidating monooxygenase and copper: a gene-nutrient interaction critical to nervous system function.

Peptidylgycine alpha-amidating monooxygenase (PAM), a highly conserved copper-dependent enzyme, is essential for the synthesis of all amidated neuropeptides. Biophysical studies revealed that the binding of copper to PAM affects its structure, and cell biological studies demonstrated that the endocytic trafficking of PAM was sensitive to copper. We review data indicating that genetic reduction of PAM expression and mild copper deficiency in mice cause similar alterations in several physiological functions known to be regulated by neuropeptides: thermal regulation, seizure sensitivity, and anxiety-like behavior.

Probing the production of amidated peptides following genetic and dietary copper manipulations.

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.