Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder associated with a variety of neurologic abnormalities. This report describes the development and initial characterization of a novel murine model system in which PDC deficiency has been introduced specifically into the developing nervous system. The absence of liveborn male and a roughly 50% reduction in female offspring following induction of the X-linked mutation indicate that extensive deficiency of PDC in the nervous system leads to pre-natal lethality. Brain tissue from surviving females at post-natal days 15 and 35 was shown to have approximately 75% of wild-type PDC activity, suggesting that a threshold of enzyme activity exists for post-natal survival. Detailed histological analyses of brain tissue revealed structural defects such as disordered neuronal cytoarchitecture and neuropil fibers in grey matter, and reduced size of bundles and disorganization of fibers in white matter. Many of the histologic abnormalities resemble those found in human female patients who carry mutations in the X-linked ortholog. These findings demonstrate a requirement for PDC activity within the nervous system for survival in utero and suggest that impaired pyruvate metabolism in the developing brain can affect neuronal migration, axonal growth and cell-cell interactions. Keywords: animal models of metabolic diseases, brain development, glucose metabolism, lipid synthesis, pyruvate dehydrogenase deficiency.

Glucose-stimulated insulin secretion (GSIS) by ␤-cells requires the generation of ATP from oxidation of pyruvate as well as generation of coupling factors involving three different pyruvate cycling shuttles. The roles of several key enzymes involved in pyruvate cycling in ␤-cells have been documented using isolated islets and ␤-cell clonal lines. To investigate the role of the pyruvate dehydrogenase (PDH) complex (PDC) in GSIS, a murine model of ␤-cell-specific PDH deficiency (␤-PDHKO) was created. Pancreatic insulin content was decreased in 1-day-old ␤-PDHKO male pups and adult male mice. The plasma insulin levels were decreased and blood glucose levels increased in ␤-PDHKO male mice from neonatal life onward. GSIS was reduced in isolated islets from ␤-PDHKO male mice with about 50% reduction in PDC activity. Impairment in a glucose tolerance test and in vivo insulin secretion during hyperglycemic clamp was evident in ␤-PDHKO adults. No change in the number or size of islets was found in pancreata from 4-wk-old ␤-PDHKO male mice. However, an increase in the mean size of individual ␤-cells in islets of these mice was observed. These findings show a key role of PDC in GSIS by pyruvate oxidation. This ␤-PDHKO mouse model represents the first mouse model in which a mitochondrial oxidative enzyme deletion by gene knockout has been employed to demonstrate an altered GSIS by ␤-cells. pyruvate cycling; ␤-cell hypertrophy INSULIN SECRETION by pancreatic islet ␤-cells is influenced by a variety of effectors, with glucose being the primary and most important stimulus. Insulin secretion requires metabolism of glucose in the ␤-cell with an associated increase in total intracellular ATP and an increase in the cytosolic ATP/ADP ratio (7,18,36). Increases in the cytosolic ATP concentration result in the sequential closure of K ATP channels, depolarization of the plasma membrane, opening of voltage-sensitive L-type Ca 2ϩ channels, Ca 2ϩ influx, and insulin secretion (15, 34). The uptake of glucose and its phosphorylation to glucose 6-phosphate are catalyzed by high Km glucose transporter 2 and the rate-limiting high Km glucokinase, respectively. These "glucosensors" ensure an increase in glucose phosphorylation, since blood glucose concentrations rise after a carbohydraterich meal. Pyruvate derived from glucose is channeled predominantly into its mitochondrial metabolism because of low levels of lactate dehydrogenase activity and of the monocarboxylate transporter-1 for lactate in the plasma membrane of islets (23,24,26,34). Another link between the cytosolic and mitochondrial metabolism of glucose is the transfer of NADH via the glycerol 3-phosphate shuttle and the malate-aspartate shuttle for ATP production (8,22).Evidence shows that metabolites derived from mitochondrial metabolism of pyruvate generated primarily from glucose and to a limited extent from other nutrients (such as amino acids) play a critical role in metabolism-secretion coupling of insulin secretion by ␤-cells (24, 26). Pyruvate is converted to acetyl-CoA and...

Many synapses contain both AMPA receptors (AMPAR) and N-methyl-d-aspartate receptors (NMDAR), but their different roles in synaptic computation are not clear. We address this issue at the auditory nerve fiber synapse (called the endbulb of Held), which is formed on bushy cells of the cochlear nucleus. The endbulb refines and relays precise temporal information to nuclei responsible for sound localization. The endbulb has a number of specializations that aid precise timing, including AMPAR-mediated excitatory postsynaptic currents (EPSCs) with fast kinetics. Voltage-clamp experiments in mouse brain slices revealed that slow NMDAR EPSCs are maintained at mature endbulbs, contributing a peak conductance of around 10% of the AMPAR-mediated EPSC. During repetitive synaptic activity, AMPAR EPSCs depressed and NMDAR EPSCs summated, thereby increasing the relative importance of NMDARs. This could impact temporal precision of bushy cells because of the slow kinetics of NMDARs. We tested this by blocking NMDARs and quantifying bushy cell spike timing in current clamp when single endbulbs were activated. These experiments showed that NMDARs contribute to an increased probability of firing, shorter latency, and reduced jitter. Dynamic-clamp experiments confirmed this effect and showed it was dose-dependent. Bushy cells can receive inputs from multiple endbulbs. When we applied multiple synaptic inputs in dynamic clamp, NMDARs had less impact on spike timing. NMDAR conductances much higher than mature levels could disrupt spiking, which may explain its downregulation during development. Thus mature NMDAR expression can support the conveying of precise temporal information at the endbulb, depending on the stimulus conditions.

Pyruvate dehydrogenase (PDH) complex (PDC) deficiency is an inborn error of pyruvate metabolism causing a variety of neurologic manifestations. Systematic analyses of development of affected brain structures and the cellular processes responsible for their impairment have not been performed due to the lack of an animal model for PDC deficiency. METHODS: In the present study we investigated a murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene encoding the α subunit of PDH to study its role on brain development and behavioral studies. RESULTS: Male embryos died prenatally but heterozygous females were born. PDC activity was reduced in the brain and other tissues in female progeny compared to age-matched control females. Immunohistochemical analysis of several brain regions showed that approximately 40% of cells were PDH−. The oxidation of glucose to CO2 and incorporation of glucose-carbon into fatty acids were reduced in brain slices from 15 day-old PDC-deficient females. Histological analyses showed alterations in several structures in white and gray matters in 35 day-old PDC-deficient females. Reduction in total cell number and reduced dendritic arbors in Purkinje neurons were observed in PDC-deficient females. Furthermore, cell proliferation, migration and differentiation into neurons by newly generated cells were reduced in the affected females during pre- and postnatal periods. PDC-deficient mice had normal locomotor activity in a novel environment but displayed decreased startle responses to loud noises and there was evidence of abnormal pre-pulse inhibition of the startle reflex. CONCLUSIONS: The results show that a reduction in glucose metabolism resulting in deficit in energy production and fatty acid biosynthesis impairs cellular differentiation and brain development in PDC-deficient mice.

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