Periodic Reporting for period 3 - MacroStability (Stability and dynamics at different spatial scales: From physiology to Alzheimer's degeneration) Reporting period: 2020-10-01 to 2022-03-31 Summary of the context and overall objectives of the project Maintaining average activity within a set-point range constitutes a fundamental property of central neural circuits. However, whether and how activity set-points are regulated remains unknown. Integrating genome-scale metabolic modeling and experimental study of neuronal homeostasis, we identified mitochondrial dihydroorotate dehydrogenase (DHODH) as a regulator of activity set-points in hippocampal networks. We found that DHODH inhibitor teriflunomide stably suppressed mean firing rates via synaptic and intrinsic excitability mechanisms by modulating mitochondrial calcium buffering and spare respiratory capacity. Bidirectional activity perturbations under DHODH blockade triggered firing rate compensation, while stabilizing firing to the lower level, indicating a change in the firing rate set-point. In vivo, teriflunomide decreased CA3-CA1 synaptic transmission, CA1 mean firing rate and attenuated susceptibility to seizures even in the intractable Dravet syndrome epilepsy model. Moreover, DHODH inhibition supressed aberrant pathological CA1 activity in familial model of Alzheimer’s disease. Our results uncover mitochondria as a key regulator of activity set-points, demonstrate the differential regulation of set-points and compensatory mechanisms, and propose a new strategy to treat brain disorders associated with hyperactivity of hippocampal circuits. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Aim 1. Define the molecular architecture of a homeostatic control system underlying firing macro-stability at the level of neuronal populations. 1.1 To identify the core molecular targets that regulate metabolic network homeostasis in hippocampal circuits, we utilized genome-scale metabolic modelling. Our analysis pointed to the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) as one of the top predicted targets that transforms towards epilepsy-resistant metabolic state. Hence, we decided to experimentally study the role of DHODH. 1.2 DHODH inhibition induces a stable reduction in spontaneous firing rates. No sign of homeostatic compensation was observed following TERI application during two days of recordings as expected for typical activity-dependent perturbations. 1.3 DHODH inhibition suppresses spare mitochondrial capacity, but not ATP production. Furthermore, TERI did not affect presynaptic ATP levels, assessed by FRET measurements. 1.4 DHODH inhibition suppresses resting mitochondrial Ca2+ levels, while enhancing Ca2+ transients during spiking activity. These results suggest that DHODH inhibition facilitates mitochondrial Ca2+ buffering during spiking activity at hippocampal boutons. 1.5 DHODH inhibition stably decreases intrinsic excitability, mEPSC amplitude and frequency. TERI induced a pronounced reduction in mEPSC amplitude following 2 days of incubation and significant, but moderate, reduction in mEPSC frequency. These data indicate that DHODH inhibition does not activate typical compensatory mechanisms, leading to the sustained reduction in MFRs. 1.6 DHODH inhibition regulates MFR set point in hippocampal networks. Addition of baclofen in the presence of TERI induced a transient reduction of the MFR that was gradually corrected over a two-day period. The adaption stabilized at the new, lower steady-state level established following TERI application. The lower set-point was re-established in each network according to its own steady-state, as well as on average across all the experiments. Importantly, partial mitochondrial uncoupling by low concentrations of Bam15 (1 µM) caused a stable reduction in the MFR, but impaired MFR renormalization following baclofen application. These experiments indicate that inhibition of specific, DHODH-dependent mitochondrial functions is critical for lowering MFR set-point without impairing homeostatic feedback responses.1.7 DHODH inhibition reduces MFR in the hippocampus in vivo. Given a profound difference in energy metabolism between in vivo and ex vivo central neuronal networks, we asked whether DHODH inhibition modulates spiking activity in vivo. We recorded single-unit activity in behaving adult mice with chronically implanted tetrodes. Compared to baseline, i.c.v. TERI injection caused a ~60% stable decrease in the MFR of CA1 neurons. Aim 2. Exploring the mechanisms leading to destabilization of neural circuits' activity associated with Alzheimer's disease.2.1 We have recently proposed that dysregulation of firing stability in hippocampal circuits and imbalance between firing stability and synaptic plasticity represent a major cause of memory impairments in early AD (Styr and Slutsky, Nature Neuroscience, 2018, Frere and Slutsky, Neuron 2018). We started the screening of fAD mutations in order to test whether they impair the basic homeostatic control system underlying stabilization of spiking rates and patterns in response to perturbations. Our results suggest that fAD mutations in either APP or PS1 proteins, impaired MFR homeostatic response of hippocampal network to hyperactivity, but not to inactivity. 2.2 DHODH inhibition rescues synaptic hyperactivity in CA3-CA1 connections of fAD mouse model. 2.2 DHODH inhibition reduces the susceptibility to seizures in Dravet syndrome model. TERI-treated group displayed 1.8-fold reduction in the frequency of interictal spikes and decreased the susceptibility for thermally-induced seizures in DS mice. Aim 3. Determining the basic principles stabilizing activity of hippocampal circuits in vivo.3.1 Establishing an integrated system for imaging of neuronal activity in the CA1 hippocampus in behaving miceUtilizing highly-sensitive GCaMP6f sensor for somatic Ca2+ imaging reflecting spiking activity, we established system for measuring neuronal Ca dynamics in the hippocampus of behaving mice based on miniaturized integrated head-mounted microscope in freely behaving mice. We have recently established this technique in my lab and demonstrated that it enables detection of stable population-level mean Ca2+ activity rate in the CA1 pyramidal neurons of mice running in a familiar environment, during a week of recording. Furthermore, we demonstrated that CA1 activity set points are regulated by arousal levels in wild-type mice. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our project is at the crossroad of several disciplines, including optical imaging, electrophysiology, big data analysis and molecular biology. The new techniques and tools developed here will be (and already) useful for other biological disciplines, such as cell biology, metabolism and neurodegenerative disorders.We established Ca imaging in the hippocampus of behaving mice using microendoscopy. The miniaturized microscope allows for high-speed and large-scale longitudinal recordings of Ca2+ dynamics from defined neuronal populations in various deep brain structures in freely behaving mice. Importantly, this technique enables detection of activity in large neuronal populations, as well as the longitudinal analysis of the same cells over weeks. Both of these are very hard to achieve using classical electrophysiological recordings, but they are essential for the questions we ask. Graphical abstract
