Motor neuropathy-associated mutation impairs Seipin functions in neurotransmission.

Gain-of-toxic-function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N-glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N-glycosylation of Seipin on synaptic transmission by over-expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over-expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post-synaptic currents and miniature inhibitory post-synaptic currents. The amplitude of both evoked excitatory post-synaptic currents and inhibitory post-synaptic current is also compromised by mutant Seipin over-expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over-expressing Seipin-N88S, whereas neither γ-amino butyric acid (GABA) nor α-Amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin-N88S-expressing neurons. These data demonstrate that Seipin-N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.

Seipin regulates excitatory synaptic transmission in cortical neurons.

Heterozygosity for missense mutations in Seipin, namely N88S and S90L, leads to a broad spectrum of motor neuropathy, while a number of loss-of-function mutations in Seipin are associated with the Berardinelli-Seip congenital generalized lipodystrophy type 2 (CGL2, BSCL2), a condition that is characterized by severe lipoatrophy, insulin resistance, and intellectual impairment. The mechanisms by which Seipin mutations lead to motor neuropathy, lipodystrophy, and insulin resistance, and the role Seipin plays in central nervous system (CNS) remain unknown. The goal of this study is to understand the functions of Seipin in the CNS using a loss-of-function approach, i.e. by knockdown (KD) of Seipin gene expression. Excitatory post-synaptic currents (EPSCs) were impaired in Seipin-KD neurons, while the inhibitory post-synaptic currents (IPSCs) remained unaffected. Expression of a shRNA-resistant human Seipin rescued the impairment of EPSC produced by Seipin KD. Furthermore, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced whole-cell currents were significantly reduced in Seipin KD neurons, which could be rescued by expression of a shRNA-resistant human Seipin. Fluorescent imaging and biochemical studies revealed reduced level of surface AMPA receptors, while no obvious ultrastructural changes in the pre-synapse were found. These data suggest that Seipin regulates excitatory synaptic function through a post-synaptic mechanism.

Neonatal overnutrition in mice exacerbates high-fat diet-induced metabolic perturbations.

Neonatal overnutrition results in accelerated development of high-fat diet (HFD)-induced metabolic defects in adulthood. To understand whether the increased susceptibility was associated with aggravated inflammation and dysregulated lipid metabolism, we studied metabolic changes and insulin signaling in a chronic postnatal overnutrition (CPO) mouse model. Male Swiss Webster pups were raised with either three pups per litter to induce CPO or ten pups per litter as control (CTR) and weaned to either low-fat diet (LFD) or HFD. All animals were killed on the postnatal day 150 (P150) except for a subset of mice killed on P15 for the measurement of stomach weight and milk composition. CPO mice exhibited accelerated body weight gain and increased body fat mass prior to weaning and the difference persisted into adulthood under conditions of both LFD and HFD. As adults, insulin signaling was more severely impaired in epididymal white adipose tissue (WAT) from HFD-fed CPO (CPO-HFD) mice. In addition, HFD-induced upregulation of pro-inflammatory cytokines was exaggerated in CPO-HFD mice. Consistent with greater inflammation, CPO-HFD mice showed more severe macrophage infiltration than HFD-fed CTR (CTR-HFD) mice. Furthermore, when compared with CTR-HFD mice, CPO-HFD mice exhibited reduced levels of several lipogenic enzymes in WAT and excess intramyocellular lipid accumulation. These data indicate that neonatal overnutrition accelerates the development of insulin resistance and exacerbates HFD-induced metabolic defects, possibly by worsening HFD-induced inflammatory response and impaired lipid metabolism.