BackgroundDown's syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology.Methodology/Principal FindingsUsing a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes.Conclusions/SignificanceThese results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.

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Increased levels of extracellular excitatory amino acids and failure of energy metabolism are two conditions associated with brain ischemia. In the present study we have combined the simultaneous inhibition of glutamate uptake and mitochondrial electron transport chain to simulate neuronal damage associated with brain ischemia. Results show that cerebellar granule neurons are not vulnerable to transient glutamate uptake inhibition by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) despite the increase in the extracellular concentration of glutamate, unless they are simultaneously exposed to the mitochondrial toxins 3-nitropropionic acid (3-NP) or sodium azide. Cell damage was assessed by light microscopy observation, by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and by the fluorescent markers for live and dead cells, calcein and ethidium homodimer, respectively. The protective effect of alternative energy substrates, such as pyruvate, acetoacetate, and beta-hydroxybutyrate against PDC-induced neuronal death during 3-NP exposure was studied and compared to the effects of the antioxidant vitamin E, the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN), voltage-dependent calcium channel antagonists, and glutamate receptor antagonists. Results show that neuronal damage can be efficiently prevented in the presence of pyruvate and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, whereas the non-NMDA receptor antagonist NBQX, acetoacetate, vitamin E, and PBN showed partial protection. In contrast, beta-hydroxybutyrate and voltage-dependent calcium channels blockers did not show any protective effect at the concentrations tested.

The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.

It is thought that the combination of extracellular glutamate accumulation and mitochondrial damage is involved in neuronal death associated with brain ischemia and hypoglycemia, and some neurodegenerative diseases such as Huntington's disease. However, the mechanism whereby those two factors interact together to trigger neurodegeneration in this and other neurodegenerative disorders is still elusive. Here, we have addressed this issue using a model of mild and sustained accumulation of extracellular glutamate in cerebellar cultured neurons, which are mostly glutamatergic and commonly used to study glutamate neurotoxicity. The resulting stimulation of glutamate receptors triggered a 50% persistent increase in mitochondrial respiration that was associated with free radicals formation, and which was found to be necessary to prevent the collapse of the mitochondial membrane potential (Dw m ) and apoptotic cell death. In fact, hampering the glutamate-mediated increase in mitochondrial respiration with an inhibitor of the mitochondrial respiratory chain stopped neurons from producing free radicals, but led them to undergo rapid and profound Dw m collapse and apoptotic cell death. Thus, we suggest that the formation of reactive oxygen species by glutamate receptor activation is the unavoidable consequence of an increase in the mitochondrial respiration aimed to prevent Dw m collapse and neurodegeneration. These results may be relevant to understand the pathophysiology of those neurodegenerative diseases associated with both mitochondrial respiratory chain and glutamate transporter defects.

SUMMARYThis 23-month-old white boy was first admitted to Jackson Memorial Hospital, Miami, Florida, at one day of age. He had been the product of a normal pregnancy and delivery, and weighed 3.97 kg at birth. At another hospital he was noted to be cyanotic and heart murmur was heard. A sibling had died of heart failure at two weeks of age. On admission the patient was noted to be cyanotic only when cry- From

An increased concentration of extracellular glutamate is associated with neuronal damage induced by cerebral ischemia. We have demonstrated previously that exposure of cultured cerebellar granule neurons to L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a glutamate uptake inhibitor, increases extracellular glutamate levels but does not induce neuronal damage. Coincubation of PDC, however, with a subthreshold concentration of the mitochondrial toxin, 3-nitropropionic acid (3-NP), results in severe damage to these neurons. We have investigated the time course of changes in mitochondrial reducing capacity and ATP levels in cerebellar granule cells after simultaneous exposure to 3-NP and PDC, and its relation to cell viability and nuclear condensation. Although individually, 3-NP and PDC treatments are not harmful to neurons, the simultaneous exposure to both compounds results in a progressive decline in mitochondrial reducing capacity during the first 4 hr, and a rapid decrease in ATP levels. At 4 hr, cells lose plasma membrane integrity and show condensed nuclei. In the presence of the energy substrates pyruvate and acetoacetate, the N-methyl-D-apartate (NMDA) receptor antagonist, MK-801, and the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN), the decline in mitochondrial activity and ATP levels is prevented, the number of condensed nuclei is reduced, and plasma membrane integrity is preserved. In contrast, the broad-spectrum caspase inhibitor Z-Asp-DCB (Z-Asp-CH2-DCB) prevents nuclear condensation but has no effect on mitochondrial reducing capacity or cell survival. Our results show that glutamate uptake impairment rapidly induces neuronal death during inhibition of succinate dehydrogenase by a mechanism involving mitochondrial dysfunction that, if not prevented, leads to cell death.

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