Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity

Global neuroprotection of huntingtin in culture and alterations of cortico-striatal connections in Huntington’s disease culture and mouse models

Huntington’s disease (HD) is a genetic neurodegenerative disorder caused by expansion of a CAG repeat in exon 1 of the HTT gene, encoding an elongated poly-glutamine repeat in the N-terminal region of the protein huntingtin (mutant huntingtin; mHtt). The average age of onset is 38, and the disease is characterized by psychiatric disorders and cognitive deficits that, in general, gradually develop over 10 years before the overt onset of the disease phenotype – difficulties in movement control. In the past two decades, many studies have focused on cell death that is obvious in mid to late stage of the disease when the overt disease symptoms become irreversible, despite the fact that altered neuronal/synaptic functions may underlie the mood/cognitive disorders that precede a motor diagnosis. In order to uncover the potentially preventable and/or reversible changes in cortico-striatal (C-S) connections in pre- and early stages of HD, we first studied the C-S coculture platform that represents its in vivo counterparts in order to investigate the role of wild-type huntingtin (wtHtt) protein in cell-death and C-S synaptic malfunctions in HD. I found that coculture with low cortical-to-striatal neuronal plating ratio (1:3 plating ratio) is a closer replica of its in vivo origin with slight differences in membrane properties, but with a significant increase in extrasynaptic NMDA receptor portion and a decrease in cell-survival signaling compared with the control (1:1). On the other hand, we found that wtHtt provides neuroprotective effects to striatal, cortical and hippocampal neurons, in a phospho-CREB-independent way in the case of the latter two neuronal types. Finally, using the C-S coculture and acute brain slice to study C-S synapse development and functions, I found that mHtt impairs the connection not only via suppressing striatal dendritic tree development but also by altering excitatory presynaptic vesicle release and recovery of the glutamate pool. In summary, this work is a further proof of HD as synaptopathy, and is a foundation for future research of drug discovery for HD targeting synaptic malfunctions at the pre-symptomatic stage.Medicine, Faculty ofGraduat

Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of Huntington disease

Background: Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the huntingtin (HTT) gene, leading to selective and progressive neuronal death predominantly in the striatum. Mutant HTT expression causes dysfunctional cortico-striatal (CS) transmission, loss of CS synapses, and striatal medium spiny neuron (MSN) dendritic spine instability prior to neuronal death. Co-culturing cortical and striatal neurons in vitro promotes the formation of functional CS synapses and is a widely used approach to elucidate pathogenic mechanisms of HD and to validate potential synapto-protective therapies. A number of relevant in vivo synaptic phenotypes from the YAC128 HD mouse model, which expresses full-length transgenic human mutant HTT, are recapitulated in CS co-culture by 21 days in vitro (DIV). However, striatal spine loss, which occurs in HD patients and in vivo animal models, has been observed in YAC128 CS co-culture in some studies but not in others, leading to difficulties in reproducing and interpreting results. Here, we investigated whether differences in the relative proportion of cortical and striatal neurons alter YAC128 synaptic phenotypes in this model. Results: YAC128 MSNs in 1:1 CS co-culture exhibited impaired dendritic length and complexity compared to wild-type, whereas reducing cortical input using a 1:3 CS ratio revealed a dramatic loss of YAC128 MSN dendritic spines. Chimeric experiments determined that this spine instability was primarily cell autonomous, depending largely on mutant HTT expression in striatal neurons. Moreover, we found that spontaneous electrophysiological MSN activity correlated closely with overall dendritic length, with no differences observed between genotypes in 1:3 co-cultures despite significant YAC128 spine loss. Finally, limiting cortical input with a 1:3 CS ratio impaired the basal survival of YAC128 neurons at DIV21, and this was partially selective for dopamine- and cAMP-regulated phosphoprotein 32-positive MSNs. Conclusions: Our findings reconcile previous discordant reports of spine loss in this model, and improve the utility and reliability of the CS co-culture for the development of novel therapeutic strategies for HD.Medicine, Faculty ofOther UBCNon UBCPsychiatry, Department ofReviewedFacult

Additional file 1: of Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of Huntington disease

Individual data values. Individual values for data with n < 6. (XLSX 12 kb

Additional file 4: of Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of Huntington disease

Figure S3. Reduced dendritic length and complexity in co-cultured YAC128 MSNs are developmental phenotypes. WT and YAC128 co-cultures were generated at either a 1:1 or 1:3 CS ratio and processed at DIV14, 18, and 21 for DARPP32 immunocytochemistry, imaging, and dendritic analysis. (A) Sample images of MSN dendritic traces generated in NeuronStudio (scale bar = 15 μm). A developmental increase in (Bi, Bii, Biii) dendritic complexity by Sholl analysis and (Biv) total dendritic length are impaired after DIV14 in co-cultured YAC128 MSNs compared to WT. Post-hoc statistical significance for Sholl analysis is shown only for WT 1:1 vs. YAC128 1:1 (*) or WT 1:3 vs. YAC128 1:3 (#) comparisons [n = 32(4); two-way ANOVA with Bonferroni post-hoc analysis; *p < 0.05, **p < 0.01, ***p < 0.001]. (TIF 875 kb

Additional file 5: of Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of Huntington disease

Figure S4. Increased thin spine density and reduced mushroom spine head diameter in DIV21 YAC128 cortical neurons. WT and YAC128 pure cortical cultures were fixed at DIV21 and subjected to in vitro DiI DiOlistic dye labeling for spine analysis. (A) Sample images of DiI-stained spines on cortical dendrites (scale bar = 5 μm). No significant differences in (Bi) total, (Bii) mushroom, or (Biii) stubby spine densities were observed in YAC128 cortical neurons. (Biv) Increased thin spine density and (Bv) reduced mushroom spine head diameter were measured in YAC128 cortical neurons compared to WT [n = 30(3); Student’s t test; *p < 0.05]. (TIF 288 kb

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity