Epilepsy Research Now in 3D: Harnessing the Power of Brain Organoids in Epilepsy

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

Discovery of Potential Anti-Ischemic Stroke Agents Through Inhibiting Sulfonylurea Receptor 1 (SUR1): A Pharmacophore-based Screening, Docking, and Molecular Dynamic Simulation

Stroke is the leading cause of disability and death worldwide. Inhibition of sulfonylurea 1 receptor (SUR1) using glibenclamide previously has been studied in CNS ischemic tissues and faster recovery from stroke injury in different animal models of stroke. Unfortunately, glibenclamide cannot enter the brain through an intact brain membrane (BBB) due to its ionization at physiological pH. Therefore, it was hypothesized that compounds with structural properties similar to glibenclamide but with the ability to penetrate through BBB would be superior to glibenclamide in ischemic stroke. Docking energy and interactions of glibenclamide with SUR1 active site were assessed using AutoDock Vina. NCI databases search engines with limitations for penetration to CNS were used to find the best compounds with desired properties. Then two selected compounds were assessed with dynamic molecular studies. Two compounds called CID-415537 and CID-419074 with docking energies of -10.3 kcal/mol and -11 kcal/mol were identified. CID-415537 was selected as the best compound due to its proper interactions with SUR1 amino acids and stability in molecular dynamic simulation. Based on this study, compound CID-415537 would be a good candidate for a SUR1 inhibitor in ischemic stroke. However, further in vivo investigations are required to confirm these findings.

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We have developed a method that through a combination of virus injections allows us to target ≤4 hippocampal cells 48% of the time and a single cell 16% of the time in wildtype mice without the use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterised in vivo. By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells.

MEG source imaging detects optogenetically-induced activity in cortical and subcortical networks

Magnetoencephalography measures neuromagnetic activity with high temporal, and theoretically, high spatial resolution. We developed an experimental platform combining MEG-compatible optogenetic techniques in nonhuman primates for use as a functional brain-mapping platform. Here we show localization of optogenetically evoked signals to known sources in the superficial arcuate sulcus of cortex and in CA3 of hippocampus at a resolution of 750 µm3. We detect activation in subcortical, thalamic, and extended temporal structures, conforming to known anatomical and functional brain networks associated with the respective sites of stimulation. This demonstrates that high-resolution localization of experimentally produced deep sources is possible within an intact brain. This approach is suitable for exploring causal relationships between discrete brain regions through precise optogenetic control and simultaneous whole brain MEG recording with high-resolution magnetic source imaging (MSI).

Rhes protein transits from neuron to neuron and facilitates mutant huntingtin spreading in the brain.

Rhes (RASD2) is a thyroid hormone-induced gene that regulates striatal motor activity and promotes neurodegeneration in Huntington disease (HD) and tauopathy. Previously, we showed that Rhes moves between cultured striatal neurons and transports the HD protein, polyglutamine-expanded huntingtin (mHTT) via tunneling nanotube (TNT)-like membranous protrusions. However, similar intercellular Rhes transport has not yet been demonstrated in the intact brain. Here, we report that Rhes induces TNT-like protrusions in the striatal medium spiny neurons (MSNs) and transported between dopamine-1 receptor (D1R)-MSNs and D2R-MSNs of intact striatum and organotypic brain slices. Notably, mHTT is robustly transported within the striatum and from the striatum to the cortical areas in the brain, and Rhes deletion diminishes such transport. Moreover, we also found transport of Rhes to the cortical regions following restricted expression in the MSNs of the striatum. Thus, Rhes is a first striatum-enriched protein demonstrated to move and transport mHTT between neurons and brain regions, providing new insights on interneuronal protein transport in the brain.

LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

The Parkinson′s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.

Unique properties of dually innervated dendritic spines in pyramidal neurons of the somatosensory cortex uncovered by 3D correlative light and electron microscopy

Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

Epigenetic MRI: Noninvasive Imaging of DNA Methylation in the Brain

It has been recognized that an integration of neuronal and genetic mechanisms supports brain function, regulates behaviour, and underpins response to environmental or disease stimuli. Several different technologies are available for imaging and studying neuronal activity in living brains, such as functional magnetic resonance imaging (fMRI), and have been translated to humans. However, the tools available to measure gene expression are destructive. Here we present a method, called epigenetic MRI (eMRI), that overcomes this limitation. eMRI achieves for the first time direct and noninvasive imaging of DNA methylation, a major gene expression regulator, in intact brains. eMRI exploits the methionine metabolic pathways that are responsible for DNA methylation to label the methyl-cytosine in brain genomic DNA through carbon-13 enriched diets. It then uses a novel carbon-13 magnetic resonance spectroscopic imaging (13C-MRSI) method to map the spatial distribution of labeled DNA. We demonstrated successful 13C labeling of brain DNA through diet using mass spectrometry, and robust and specific detection of labeled DNA using 13C-MRSI. We used eMRI and a biomedical piglet model to produce the first DNA methylation map of an intact brain hemisphere. With both noninvasive labeling and imaging, we expect eMRI to be readily translated to humans and thus enable many new investigations into the epigenetic basis of brain function, behavior, and disease.

Direct focus sensing and shaping for high-resolution multi-photon imaging in deep tissue

High-resolution optical imaging of deep tissue in-situ such as the living brain is fundamentally challenging because of the aberration and scattering of light. In this work, we develop an innovative adaptive optics three-photon microscope based on direct focus sensing and shaping that can accurately measure and effectively compensate for both low- and high-order specimen-induced aberrations and recover near-diffraction-limited performance at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 um below pia, making high-resolution microscopy in cortex near non-invasive. Functional calcium imaging with high sensitivity and accuracy, and high-precision laser-mediated microsurgery through the intact skull were demonstrated. Moreover, we also achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below pia within the intact brain.