Pathogenic Variants in GPC4 Cause Keipert Syndrome.

Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.

Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

A preferential role for glycolysis in preventing the anoxic depolarization of rat hippocampal area CA1 pyramidal cells.

During brain anoxia or ischemia, a decrease in the level of ATP leads to a sudden decrease in transmembrane ion gradients [anoxic depolarization (AD)]. This releases glutamate by reversing the operation of glutamate transporters, which triggers neuronal death. By whole-cell clamping CA1 pyramidal cells, we investigated the energy stores that delay the occurrence of the AD in hippocampal slices when O2 and glucose are removed. With glycolytic and mitochondrial ATP production blocked in P12 slices, the AD occurred in approximately 7 min at 33 degrees C, reflecting the time needed for metabolic activity to consume the existing ATP and phosphocreatine, and for subsequent ion gradient decrease. Allowing glycolysis fueled by glycogen, in the absence of glucose, delayed the AD by 5.5 min, whereas superfused glucose prevented the AD for >1 h. With glycolysis blocked, the latency to the AD was 6.5 min longer when mitochondria were allowed to function, demonstrating that metabolites downstream of glycolysis (pyruvate, citric acid cycle intermediates, and amino acid oxidation) provide a significant energy store for oxidative phosphorylation. With glycolysis blocked but mitochondria functioning, superfusing lactate did not significantly delay the AD, showing that ATP production from lactate is much less than that from endogenous metabolites. These data demonstrate a preferential role for glycolysis in preventing the AD. They also define a hierarchy of pool sizes for hippocampal energy stores and suggest that brain ATP production from glial lactate may not be significant in conditions of energy deprivation.