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.

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 α-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxietylike 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.

The mammalian amygdala expresses various neuropeptides whose signaling has been implicated in emotionality. Many neuropeptides require amidation for full activation by peptidylglycine ␣-amidating monooxygenase (PAM), a transmembrane vesicular cuproenzyme and regulator of the secretory pathway. Mice heterozygous for the Pam gene (PAM ϩ/Ϫ ) 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 ϩ/Ϫ amygdala. PAM ϩ/Ϫ mice presented with anxiety-like behaviors in the zero maze that were alleviated by diazepam. PAM ϩ/Ϫ 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 ϩ/Ϫ amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM ϩ/Ϫ 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 ϩ/Ϫ lateral nucleus. Phasic GABAergic signaling was also augmented in the PAM ϩ/Ϫ amygdala, and this difference comprised activity-independent and -dependent components. These physiological findings represent perturbations in the PAM ϩ/Ϫ amygdala that may underlie the aberrant emotional responses in the intact animal.

Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

Peptidylgycine α-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.