Lentiviral Transgenesis as a Tool to Study the Role of Uncoupling Protein Isoforms in the Nervous System

A tight coupling exists between synaptic activity and glucose utilization by astrocytes. Metabolic cooperation between neurons and astrocytes mediates this coupling. During synaptic activation, glutamate that is released in the synaptic cleft as a neurotransmitter by neurons is rapidly cleared by an active uptake into astrocytes. One glutamate is co-transported with three Na+. Intracellular astrocytic Na+ homeostasis is re-established by the Na+/K+-ATPase which requires ATP provided mainly by glycolysis. The resulting lactate is released in extracellular space and contributes to the energetic balance of activated neurons. This coupling between astrocytes and neurons is known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH). The role of mitochondria in this coupling remains to be determined and more specifically the role of uncoupling proteins (UCP) which have been recently identified in neural tissues. UCPs belong to a family of mitochondrial carriers which are present in the inner mitochondrial membrane. There are five isoforms named UCP1 to UCP5. The well-known characterized is UCP1, also named thermogenin, which is present in the brown adipose tissue (BAT) of the small rodents and is responsible of non-shivering thermogenesis. It dissipates the proton gradient across the inner mitochondrial membrane, thereby producing heat instead of ATP. UCP2 is ubiquitous and is implicated in protection against excessive reactive oxygen (ROS) production. The expression of UCP3 is restricted to skeletal muscle where it is linked to fatty acid metabolism. The role of the brain isoforms UCP4 and UCP5 is still poorly understood. Initially, we set out to evaluate their function in energy metabolism of astrocytes and neurons. We used the lentiviral strategy to overexpress or silence the UCPs isoforms in primary cultures of astrocytes and neurons. We found that only UCP4 and UCP5 had an uncoupling activity in brain cells. Indeed, we observed a reduced mitochondrial potential and a reduced ATP/ADP ratio in astrocytes as well as in neurons overexpressing UCP4 or UCP5. UCP4 reduced the oxidative pathway of astrocytes without modifying the basal glucose metabolism and without having a lethal effect on the cell. Oxygen consumption rate (OCR) of brain cells ovexpressing UCPs was reduced. Moreover, UCP4 increased lactate release by astrocytes, suggesting an implication in the astrocyte-neuron coupling. We also brought evidence that both UCP4 and UCP5 partially protect brain cells from oxidative damage. All these results were confirmed by experiments on UCPs silencing. In a second step, we used a co-culture model to study the impact of astrocytic uncoupling protein on neuronal survival and we demonstrated that the presence of uncoupling protein in astrocytes prevents neuronal death after glutamate excitotoxicity. Taken together, all these results support an important role of uncoupling proteins in the regulation of astrocytic energy production, thus promoting local export of lactate which can be used by neurons.

Deciphering the astrocyte-neuron metabolic cooperation in vivo

Manuel Raphael Zenger

Brain energy consumption is high and a very tightly regulated energy metabolism ensures correct performance. Neurons, the cells responsible for transmitting information via synapses, interact very closely with star shaped glial cells called astrocytes. Astrocytes play a pivotal role in the regulation of brain energy metabolism and for neurotransmission. Especially, in the case of glutamate, the main excitatory neurotransmitter in the brain, astrocytes are contributing to the clearing of otherwise potentially toxic levels of glutamate in the synaptic cleft. In addition, astrocytes are capable of providing neurons with lactate, which can be used as an additional source of energy in periods of increased demand, such as during neurotransmission. Lactate released by astrocytes is also a signal for neuronal plasticity. In this work, we addressed different aspects of the astrocyte-neuron metabolic coupling. Uncoupling proteins (UCPs) are proteins of the inner mitochondrial membrane that are capable of dissipating a proton gradient (uncoupling ATP production from respiration). Five different UCP isoforms have been described. UCP1 is essentially found in brown adipose tissue and is implicated in non-shivering thermogenesis, UCP2 is widely expressed and protecting against reactive oxygen species and UCP3 is mainly expressed in skeletal muscle tissue where a possible role in fatty acid metabolism has been described. UCP4 and 5 are essentially expressed in the brain, but their precise function still needs to be determined. In the present study, we showed that in neurons, UCP4 and UCP5 are predominantly expressed, as compared to the other UCPs. In astrocytes, UCP5 is the most abundant isoform, but also UCP2 and 4 are significantly expressed indicating an important role for these three UCPs in astrocytes. The role of UCP4 was further elucidated. For this purpose, a lentiviral overexpressing UCP4 construct was produced and characterized. This construct overexpressing UCP4 at the mitochondria allowed to demonstrate a neuro-protective role of UCP4 expression in astrocytes. In a complementary approach to study neuron-astrocyte metabolic coupling, the role of different astrocytic and neuronal genes in memory and learning was explored in a spatial learning paradigm. Mice were trained 1 to 9 days in a radial maze. The gene analysis showed that genes involved in lactate production and utilization such as lactate dehydrogenases were highly increased. A gene involved in the shuttling of lactate from astrocytes to neurons, such as monocarboxylate transporter 1 was also upregulated. An unexpected decrease in glucose transporter 1 and decrease in hexokinase 2 expression were observed. Finally, genes involved in glycogen metabolism were upregulated. Na+/K+-ATPase alpha2 isoform (ATP1A2), which plays a key role in re-establishing Na+ homeostasis disrupted by the co-transport of Na+ along with glutamate during glutamate uptake, was upregulated following spatial learning. Using a lentiviral transgenesis method, an attempt was made to downregulate the expression of the ATP1A2 in vivo. However, this technique did not allow us to produce sufficient knock down to study the role of ATP1A2 in vivo. Preliminary data for an alternative method are shown. Overall, the set of results presented here provides additional support for the existence of a dynamically regulated neuron-astrocyte metabolic coupling.

EPFL2015

Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization

Pierre Magistretti, Luc Pellerin

Glutamate, released at a majority of excitatory synapses in the central nervous system, depolarizes neurons by acting at specific receptors. Its action is terminated by removal from the synaptic cleft mostly via Na(+)-dependent uptake systems located on both neurons and astrocytes. Here we report that glutamate, in addition to its receptor-mediated actions on neuronal excitability, stimulates glycolysis--i.e., glucose utilization and lactate production--in astrocytes. This metabolic action is mediated by activation of a Na(+)-dependent uptake system and not by interaction with receptors. The mechanism involves the Na+/K(+)-ATPase, which is activated by an increase in the intracellular concentration of Na+ cotransported with glutamate by the electrogenic uptake system. Thus, when glutamate is released from active synapses and taken up by astrocytes, the newly identified signaling pathway described here would provide a simple and direct mechanism to tightly couple neuronal activity to glucose utilization. In addition, glutamate-stimulated glycolysis is consistent with data obtained from functional brain imaging studies indicating local nonoxidative glucose utilization during physiological activation.

1994

Elucidating the role of neuron-astrocyte metabolic coupling in learning and memory

Monika Tadi

The brain has very high energy demands that are mainly met by the circulating blood glucose to ensure its proper functioning. Thus, it is not surprising that though the human brain weighs only 2- 3% of the body weight, it consumes approximately 25% of total body glucose. “Neuro-energetic coupling" is a unique feature of the brain and refers to the existence of the tight coupling between (neuronal) synaptic brain activity and energy metabolism. The main excitatory neurotransmitter in the brain is glutamate and most of the energy requirements at the cortical level are met through glutamate-mediated neurotransmission, suggesting that there is a close relationship between brain activity, glutamatergic neurotransmission and energy metabolism. Until the 80s, blood-borne glucose was thought to be the sole energy substrate of the brain cells and it was assumed that glucose is metabolized by neurons and astrocytes (a type of glial cell) independently. It is only during the last two decades that the concept of compartmentalization of energy metabolism between neurons and astrocytes has become more obvious. The transfer of lactate from astrocytes to neurons termed as the “astrocyte-neuron lactate shuttle (ANLS)” model proposed by Dr. Pellerin and Prof. Magistretti posits that glutamate released during brain activation activates glycolysis (one of glucose metabolic pathways) in astrocytes leading to lactate (a monocarboxylate) release in the extracellular space, and this astrocytic lactate is then captured and used as an energy substrate by the activated neurons to meet their energy requirements. It includes a complex chain of events involved in neuro-metabolic coupling and supports the preferential use of lactate by activated neurons under conditions of increased energy demands. Higher brain functions such as learning a new task are associated with structural changes in brain and are metabolically demanding necessitating the need of an additional energy supply to meet the neurons increased energy requirement. In this context, the current thesis project aimed to elucidate the contribution of metabolic coupling between astrocytes and neurons, known as “neuron-astrocyte metabolic coupling” to learning and memory formation. Using an in vivo approach combined with behavioral, molecular and gene manipulation techniques in mice, we set out to investigate the role of neuron-astrocyte metabolic coupling, particularly ANLS in cognition. The project was divided into two main parts: In the first part, the expression of glucose metabolism-related genes was investigated during long term memory (LTM) formation in fear-motivated inhibitory avoidance (IA) learning paradigm. Using (14C) 2-Deoxyglucose (2-DG) technique, we first defined the brain areas that are involved in IA LTM formation. This technique provides an indirect measurement of brain activity by measuring glucose uptake in different brain areas during the task. The results of brain metabolic mapping show an increase in glucose utilization in the hippocampus, amygdala, anterior cingulate cortex and pre-limbic and infra-limbic cortical areas. Results from the quantitative analysis of mRNA levels in the dorsal hippocampus at different time point’s following IA task highlight a time dependent, learning specific change in the expression of genes related to glucose metabolism. Specific set of genes involved in the synthesis and degradation of glycogen (brain glucose reserve present only in astrocytes), pyruvate metabolism and pentose phosphate shunt as well as the ANLS were induced following the IA task. These early observations indicate that the metabolic interactions between astrocytes and neurons undergo modifications during a learning process and suggest that these adaptations may play a key role in the establishment of long-term memory. In the second part of research project, we focused on investigating the behavioral consequences of down regulating the expression of Monocarboxylate Transporter 1 (MCT1), a key ANLS related gene, on cognition and higher brain functions. Mice heterozygous for the MCT1 gene (MCT1+/-) were characterized in numerous behavioral paradigms such as general wellbeing, reflexive and motor capabilities. After having established that MCT1+/- mice have no alterations in general emotional state or in locomotion, a battery of behavioral tests was performed to study cognition in these mice. Compared with wild-type control mice, the MCT1+/- mice display normal muscle strength and motor coordination. However, when tested for higher brain functions, the MCT1+/- mice exhibit learning and memory deficits in several learning paradigms/tasks that are hippocampus and / or amygdala dependent. The cognitive impairment observed in the MCT1 heterozygous mice further highlights the importance of ANLS in memory and suggest that disruption of lactate transfer from astrocytes to neurons via the MCT1 results in cognitive impairment. The overall results in this thesis demonstrate that cerebral energy metabolism, and in particular the transfer of lactate from astrocytes to neurons not only provides temporal adaptations in the learning process, but it also plays a fundamental role in memory formation.