Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

Information processing is energetically expensive. In the mammalian brain, it is unclear how information coding and energy usage are regulated during food scarcity. I addressed this in the visual cortex of awake mice using whole-cell recordings and two-photon imaging to monitor layer 2/3 neuronal activity and ATP usage. I found that food restriction reduced synaptic ATP usage by 29% through a decrease in AMPA receptor conductance. Neuronal excitability was nonetheless preserved by a compensatory increase in input resistance and a depolarized resting membrane potential. Consequently, neurons spiked at similar rates as controls, but spent less ATP on underlying excitatory currents. This energy-saving strategy had a cost since it amplified the variability of visually-evoked subthreshold responses, leading to a 32% broadening in orientation tuning and impaired fine visual discrimination. This reduction in coding precision was associated with reduced levels of the fat mass-regulated hormone leptin and was restored by exogenous leptin supplementation. These findings reveal novel mechanisms that dynamically regulate energy usage and coding precision in neocortex.

Conventionally, neurons are thought to be cellular units that process synaptic inputs into synaptic spikes. However, it is well known that neurons can also spike spontaneously and display a rich repertoire of firing properties with no apparent functional relevance e.g. in in vitro cortical slice preparations. In this talk, I will propose a hypothesis according to which intrinsic excitability in neurons may be a survival mechanism to minimize toxic byproducts of the cell’s energy metabolism. In neurons, this toxicity can arise when mitochondrial ATP production stalls due to limited ADP. Under these conditions, electrons deviate from the electron transport chain to produce reactive oxygen species, disrupting many cellular processes and challenging cell survival. To mitigate this, neurons may engage in ADP-producing metabolic spikes. I will explore the validity of this hypothesis using computational models that illustrate the implications of synaptic and metabolic spiking, especially in the context of substantia nigra pars compacta dopaminergic neurons and their degeneration in Parkinson's disease.

Information processing is energetically expensive. In the mammalian brain, it is unclear how information coding and energy usage are regulated during food scarcity. We addressed this in the visual cortex of awake mice using whole-cell patch clamp recordings and two-photon imaging to monitor layer 2/3 neuronal activity and ATP usage. We found that food restriction resulted in energy savings through a decrease in AMPA receptor conductance, reducing synaptic ATP usage by 29%. Neuronal excitability was nonetheless preserved by a compensatory increase in input resistance and a depolarized resting membrane potential. Consequently, neurons spiked at similar rates as controls, but spent less ATP on underlying excitatory currents. This energy-saving strategy had a cost since it amplified the variability of visually-evoked subthreshold responses, leading to a 32% broadening in orientation tuning and impaired fine visual discrimination. These findings reveal novel mechanisms that dynamically regulate energy usage and coding precision in neocortex.

Coordinated activity across networks of neurons is a hallmark of both resting and active behavioral states in many species, including worms, flies, fish, mice and humans. These global patterns alter energy metabolism in the brain over seconds to hours, making oxygen consumption and glucose uptake widely used proxies of neural activity. However, whether changes in neural activity are causally related to changes in metabolic flux in intact circuits on the sub-second timescales associated with behavior, is unclear. Moreover, it is unclear whether differences between rest and action are associated with spatiotemporally structured changes in neuronal energy metabolism at the subcellular level. My work combines two-photon microscopy across the fruit fly brain with sensors that allow simultaneous measurements of neural activity and metabolic flux, across both resting and active behavioral states. It demonstrates that neural activity drives changes in metabolic flux, creating a tight coupling between these signals that can be measured across large-scale brain networks. Further, using local optogenetic perturbation, I show that even transient increases in neural activity result in rapid and persistent increases in cytosolic ATP, suggesting that neuronal metabolism predictively allocates resources to meet the energy demands of future neural activity. Finally, these studies reveal that the initiation of even minimal behavioral movements causes large-scale changes in the pattern of neural activity and energy metabolism, revealing unexpectedly widespread engagement of the central brain.

By continuously producing electrical signals, neurones are amongst the most energy-demanding cells in the organism. Resting ionic levels are restored via metabolic pumps that receive the necessary energy from oxygen supplied by blood vessels. Intense spontaneous neural activity is omnipresent in the developing CNS. It occurs during short, well-defined periods that coincide precisely with the timing of angiogenesis. Such coincidence cannot be random; there must be a universal mechanism triggering spontaneous activity concurrently with blood vessels invading neural territories for the first time. However, surprisingly little is known about the role of neural activity per se in guiding angiogenesis. Part of the reason is that it is challenging to study developing neurovascular networks in tri-dimensional space in the brain. We investigate these questions in the neonatal mouse retina, where blood vessels are much easier to visualise because they initially grow in a plane, while waves of spontaneous neural activity (spreading via cholinergic starburst amacrine cells) sweep across the retinal ganglion cell layer, in close juxtaposition with the growing vasculature. Blood vessels reach the periphery by postnatal day (P) 7-8, shortly before the cholinergic waves disappear (at P10). We discovered transient clusters of auto-fluorescent cells that form an annulus around the optic disc, gradually expanding to the periphery, which they reach at the same time as the growing blood vessels. Remarkably, these cells appear locked to the frontline of the growing vasculature. Moreover, by recording waves with a large-scale multielectrode array that enables us to visualise them at pan-retinal level, we found that their initiation points are not random; they follow a developmental centre-to-periphery pattern similar to the clusters and blood vessels. The density of growing blood vessels is higher in cluster areas than in-between clusters at matching eccentricity. The cluster cells appear to be phagocytosed by microglia. Blocking Pannexin1 (PANX1) hemichannels activity with probenecid completely blocks the spontaneous waves and results in the disappearance of the fluorescent cell clusters. We suggest that these transient cells are specialised, hyperactive neurones that form spontaneous activity hotspots, thereby triggering retinal waves through the release of ATP via PANX1 hemichannels. These activity hotspots attract new blood vessels to enhance local oxygen supply. Signalling through PANX1 attracts microglia that establish contact with these cells, eventually eliminating them once blood vessels have reached their vicinity. The auto-fluorescence that characterises the cell clusters may develop only once the process of microglial phagocytosis is initiated.

Carnosine is a natural dipeptide widely distributed in mammalian tissues and exists at particularly high concentrations in skeletal and cardiac muscles and brain. A growing body of evidence shows that carnosine is involved in many cellular defense mechanisms against oxidative stress, including inhibition of amyloid-β (Aβ) aggregation, modulation of nitric oxide (NO) metabolism, and scavenging both reactive nitrogen and oxygen species. Different types of cells are involved in the innate immune response, with macrophage cells representing those primarily activated, especially under different diseases characterized by oxidative stress and systemic inflammation such as depression and cardiovascular disorders. Microglia, the tissue-resident macrophages of the brain, are emerging as a central player in regulating key pathways in central nervous system inflammation; with specific regard to Alzheimer’s disease (AD) these cells exert a dual role: on one hand promoting the clearance of Aβ via phagocytosis, on the other hand increasing neuroinflammation through the secretion of inflammatory mediators and free radicals. The activity of carnosine was tested in an in vitro model of macrophage activation (M1) (RAW 264.7 cells stimulated with LPS + IFN-γ) and in a well-validated model of Aβ-induced neuroinflammation (BV-2 microglia treated with Aβ oligomers). An ample set of techniques/assays including MTT assay, trypan blue exclusion test, high performance liquid chromatography, high-throughput real-time PCR, western blot, atomic force microscopy, microchip electrophoresis coupled to laser-induced fluorescence, and ELISA aimed to evaluate the antioxidant and anti-inflammatory activities of carnosine was employed. In our experimental model of macrophage activation (M1), therapeutic concentrations of carnosine exerted the following effects: 1) an increased degradation rate of NO into its non-toxic end-products nitrite and nitrate; 2) the amelioration of the macrophage energy state, by restoring nucleoside triphosphates and counterbalancing the changes in ATP/ADP, NAD+/NADH and NADP+/NADPH ratio obtained by LPS + IFN-γ induction; 3) a reduced expression of pro-oxidant enzymes (NADPH oxidase, Cyclooxygenase-2) and of the lipid peroxidation product malondialdehyde; 4) the rescue of antioxidant enzymes expression (Glutathione peroxidase 1, Superoxide dismutase 2, Catalase); 5) an increased synthesis of transforming growth factor-β1 (TGF-β1) combined with the negative modulation of interleukines 1β and 6 (IL-1β and IL-6), and 6) the induction of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1). In our experimental model of Aβ-induced neuroinflammation, carnosine: 1) prevented cell death in BV-2 cells challenged with Aβ oligomers; 2) lowered oxidative stress by decreasing the expression of inducible nitric oxide synthase and NADPH oxidase, and the concentrations of nitric oxide and superoxide anion; 3) decreased the secretion of pro-inflammatory cytokines such as IL-1β simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-β1; 4) prevented Aβ-induced neurodegeneration in primary mixed neuronal cultures challenged with Aβ oligomers and these neuroprotective effects was completely abolished by SB431542, a selective inhibitor of type-1 TGF-β receptor. Overall, our data suggest a novel multimodal mechanism of action of carnosine underlying its protective effects in macrophages and microglia and the therapeutic potential of this dipeptide in counteracting pro-oxidant and pro-inflammatory phenomena observed in different disorders characterized by elevated levels of oxidative stress and inflammation such as depression, cardiovascular disorders, and Alzheimer’s disease.

Spontaneous firing, observed in many neurons, is often attributed to ion channel or network level noise. Cortical cells during slow wave sleep exhibit transitions between so called Up and Down states. In this sleep state, with limited sensory stimuli, neurons fire in the Up state. Spontaneous firing is also observed in slices of cholinergic interneurons, cerebellar Purkinje cells and even brainstem inspiratory neurons. In such in vitro preparations, where the functional relevance is long lost, neurons continue to display a rich repertoire of firing properties. It is perplexing that these neurons, instead of saving their energy during information downtime and functional irrelevance, are eager to fire. We propose that spontaneous firing is not a chance event but instead, a vital activity for the well-being of a neuron. We postulate that neurons, in anticipation of synaptic inputs, keep their ATP levels at maximum. As recovery from inputs requires most of the energy resources, neurons are ATP surplus and ADP scarce during synaptic quiescence. With ADP as the rate-limiting step, ATP production stalls in the mitochondria when ADP is low. This leads to toxic Reactive Oxygen Species (ROS) formation, which are known to disrupt many cellular processes. We hypothesize that spontaneous firing occurs at these conditions - as a release valve to spend energy and to restore ATP production, shielding the neuron against ROS. By linking a mitochondrial metabolism model to a conductance-based neuron model, we show that spontaneous firing depends on baseline ATP usage and on ATP-cost-per-spike. From our model, emerges a mitochondrial mediated homeostatic mechanism that provides a recipe for different firing patterns. Our findings, though mostly affecting intracellular dynamics, may have large knock-on effects on the nature of neural coding. Hitherto it has been thought that the neural code is optimised for energy minimisation, but this may be true only when neurons do not experience synaptic quiescence.