Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches.

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter.

The development of fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge for noninvasive optical imaging. Here, we report the design, synthesis, and application of near-infrared (NIR)-absorbing and -emitting optical voltmeter based on a sulfonated, phosphine-oxide (po) rhodamine for voltage imaging in intact retinas. We find that po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show a range of voltage sensitivities (13 to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca2+-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca2+ imaging and traditional multielectrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity, revealing differences in voltage and Ca2+ dynamics within hyperactive networks of the mouse retina. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites.

Neurons receive a multitude of synaptic inputs along their dendritic arbor, but how this highly heterogeneous population of synaptic compartments is spatially organized remains unclear. By measuring N-methyl-d-aspartic acid receptor (NMDAR)-driven calcium responses in single spines, we provide a spatial map of synaptic calcium signals along dendritic arbors of hippocampal neurons and relate this to measures of synapse structure. We find that quantal NMDAR calcium signals increase in amplitude as they approach a thinning dendritic tip end. Based on a compartmental model of spine calcium dynamics, we propose that this biased distribution in calcium signals is governed by a gradual, distance-dependent decline in spine size, which we visualized using serial block-face scanning electron microscopy. Our data describe a cell-autonomous feature of principal neurons, where tapering dendrites show an inverse distribution of spine size and NMDAR-driven calcium signals along dendritic trees, with important implications for synaptic plasticity rules and spine function.

A Small-Molecule Photoactivatable Optical Sensor of Transmembrane Potential.

This paper discloses the design, synthesis, and imaging applications of the first member of a new class of SPOTs, small-molecule photoactivatable optical sensors of transmembrane potential. SPOT2.1.Cl features an established voltage-sensitive dye, VoltageFluor2.1.Cl--or VF--capped with a dimethoxy-o-nitrobenzyl (DMNB) caging group to effectively diminish fluorescence of the VF dye prior to uncaging. SPOT2.1.Cl localizes to cell membranes and displays weak fluorescence until photoactivated. Illumination generates the parent VF dye which then optically reports on changes in the membrane voltage. After photoactivation with spatially restricted light, SPOT2.1.Cl-loaded cells display bright, voltage-sensitive fluorescence associated with the plasma membrane, while neighboring cells remain dark. Activated SPOT reports on action potentials in single trials. SPOT can be activated in neuron cell bodies or uncaged in dendrites to enable structural tracing via "backfilling" of the dye to the soma, followed by functional imaging in the labeled cell. The combination of cellular specificity achieved through spatially defined patterns of illumination, coupled with the fast, sensitive, and noncapacitive voltage sensing characteristics of VF dyes makes SPOT2.1.Cl a useful tool for interrogating both structure and function of neuronal systems.

A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing.

This paper describes the design and synthesis of a photostable, far-red to near-infrared (NIR) platform for optical voltage sensing. We developed a new, sulfonated silicon rhodamine fluorophore and integrated it with a phenylenevinylene molecular wire to create a Berkeley Red Sensor of Transmembrane potential, or BeRST 1 ("burst"). BeRST 1 is the first member of a class of far-red to NIR voltage sensitive dyes that make use of a photoinduced electron transfer (PeT) trigger for optical interrogation of membrane voltage. We show that BeRST 1 displays bright, membrane-localized fluorescence in living cells, high photostability, and excellent voltage sensitivity in neurons. Depolarization of the plasma membrane results in rapid fluorescence increases (24% ΔF/F per 100 mV). BeRST 1 can be used in conjunction with fluorescent stains for organelles, Ca(2+) indicators, and voltage-sensitive fluorescent proteins. In addition, the red-shifted spectral profile of BeRST 1, relative to commonly employed optogenetic actuators like ChannelRhodopsin2 (ChR2), which require blue light, enables optical electrophysiology in neurons. The high speed, sensitivity, photostability and long-wavelength fluorescence profiles of BeRST 1 make it a useful platform for the noninvasive, optical dissection of neuronal activity.

Robust self-supervised denoising of voltage imaging data using CellMincer.

Voltage imaging enables high-throughput investigation of neuronal activity, yet its utility is often constrained by a low signal-to-noise ratio (SNR). Conventional denoising algorithms, such as those based on matrix factorization, impose limiting assumptions about the noise process and the spatiotemporal structure of the signal. While deep learning based denoising techniques offer greater adaptability, existing approaches fail to fully exploit the fast temporal dynamics and unique short- and long-range dependencies within voltage imaging datasets. Here, we introduce CellMincer, a novel self-supervised deep learning method designed specifically for denoising voltage imaging datasets. CellMincer operates on the principle of masking and predicting sparse sets of pixels across short temporal windows and conditions the denoiser on precomputed spatiotemporal auto-correlations to effectively model long-range dependencies without the need for large temporal denoising contexts. We develop and utilize a physics-based simulation framework to generate realistic datasets for rigorous hyperparameter optimization and ablation studies, highlighting the key role of conditioning the denoiser on precomputed spatiotemporal auto-correlations to achieve 3-fold further reduction in noise. Comprehensive benchmarking on both simulated and real voltage imaging datasets, including those with paired patch-clamp electrophysiology (EP) as ground truth, demonstrates CellMincer's state-of-the-art performance. It achieves substantial noise reduction across the entire frequency spectrum, enhanced detection of subthreshold events, and superior cross-correlation with ground-truth EP recordings. Finally, we demonstrate how CellMincer's addition to a typical voltage imaging data analysis workflow improves neuronal segmentation, peak detection, and ultimately leads to significantly enhanced separation of functional phenotypes.

Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

Optical Spike Detection and Connectivity Analysis With a Far-Red Voltage-Sensitive Fluorophore Reveals Changes to Network Connectivity in Development and Disease.

The ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential-1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis (OSCA) for high-throughput dissection of neuronal activity dynamics. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta 1-42 (Aß 1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during maturation in culture, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community.

Imaging Voltage in Complete Neuronal Networks Within Patterned Microislands Reveals Preferential Wiring of Excitatory Hippocampal Neurons.

Voltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging, post hoc immunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal ensembles. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands are similar to neurons grown on coverslips, with parallel developmental trajectories and composition of inhibitory and excitatory cell types (CA1, CA3, and dentate granule cells, or DGC). We calculate the likelihood that action potential firing in one neuron triggers action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types.

Functional imaging in the zebrafish retinotectal system using RGECO.

Genetically encoded calcium indicators (GECIs) allow repeated, non-invasive measurements of neural activity in defined populations of neurons, but until recently GECIs based on single fluorescent proteins have been limited to the green region of the color spectrum. Recent efforts in protein engineering have expanded the color palette of GECIs. One of these new GECIs, the red RGECO, is spectrally separate from the traditional GFP-based sensors such as GCaMP, and therefore opens the way for simultaneous, multicolor imaging of neural activity. While RGECO has been shown to report spontaneous calcium fluctuations in neurons, the precise relationship of RGECO signal to evoked-neural activity is not known. Measurements of neural activity using RGECO in vivo have also not been reported. Using dissociated hippocampal neurons we performed a systematic analysis of two forms of RGECO- a cytosolic form and a presynaptically localized form generated by fusion of RGECO to the presynaptic protein, synaptophysin (SyRGECO). We find that RGECO and GCaMP3 are comparable in terms of dynamic range, signal-to-noise ratios and kinetics but that RGECO is a more reliable reporter of single action potentials. In terms of performance SyGCaMP3 and SyRGECO are comparable, and both are more sensitive reporters of activity than the cytosolic form of each probe. Using the zebrafish retinotectal system we show that SyRGECO and RGECO are can report neural activity in vivo and that RGECO expression permits detailed structural analysis of neuronal arbors. We have exploited these attributes to provide a morphological and functional description of tectal cells selective for motion along the vertical axis. These results open up the possibility of using zebrafish to functionally image genetically defined pre- and postsynaptic circuit components, separable by color, which will be a powerful approach to studying neural interactions in the brain.

Parametric functional maps of visual inputs to the tectum.

How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientation-selective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientation-selective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.