Revealing the MRI-Contrast in Optically Cleared Brains.

The current consensus holds that optically-cleared specimens are unsuitable for Magnetic Resonance Imaging (MRI); exhibiting absence of contrast. Prior studies combined MRI with tissue-clearing techniques relying on the latter's ability to eliminate lipids, thereby fostering the assumption that lipids constitute the primary source of ex vivo MRI-contrast. Nevertheless, these findings contradict an extensive body of literature that underscores the contribution of other features to contrast. Furthermore, it remains unknown whether non-delipidating clearing methods can produce MRI-compatible specimens or whether MRI-contrast can be re-established. These limitations hinder the development of multimodal MRI-light-microscopy (LM) imaging approaches. This study assesses the relation between MRI-contrast, and delipidation in optically-cleared whole brains following different tissue-clearing approaches. It is demonstrated that uDISCO and ECi-brains are MRI-compatible upon tissue rehydration, despite both methods' substantial delipidating-nature. It is also demonstrated that, whereas Scale-clearing preserves most lipids, Scale-cleared brain lack MRI-contrast. Furthermore, MRI-contrast is restored to lipid-free CLARITY-brains without introducing lipids. Our results thereby dissociate between the essentiality of lipids to MRI-contrast. A tight association is found between tissue expansion, hyperhydration and loss of MRI-contrast. These findings then enabled us to develop a multimodal MRI-LM-imaging approach, opening new avenues to bridge between the micro- and mesoscale for biomedical research and clinical applications.

Datasets assessing lipid-content in optically cleared brains.

Multi-modal imaging, by light-microscopy (LM) and Magnetic Resonance Imaging (MRI), holds promise for examining the brain across various resolutions and scales. While MRI acquires images in three dimensions, acquisition of intact whole-brain by LM requires a process of tissue clearing that renders the brain transparent. Removal of lipids (delipidation) is a critical step in the tissue clearing process, and was previsouly suggested to be the cause for absence of MRI contrast in cleared brains. Yet, the association between MRI contrast, delipidation and the different clearing techniques is debatable. Here, we provide datasets concerning lipid-content in cleared brain tissues obtained by various approaches. Fixed mouse and rat brains were cleared by CLARITY, Scale, uDISCO and ECi clearing techniques. Lipid-content was assessed at various intermediate steps of the different clearing methods, as well as at the end of the processes. Methods employed included whole brain MRI acquisition, Oil Red O (ORO)- and carbocyanine DiI-staining of cryosections, and DiI-washout assay from brain slices. MRI contrast-to-noise ratio, staining intensities and integrity of tissue were systematically analyzed. We demonstrate that lipid electrophoresis, an essential step of the CLARITY approach, engenders progressive reduction in MRI contrast in non-cleared (PFA-fixed) control brains, as well as strongly reduces contrast from uDISCO and ECi-cleared brains. ORO minimally stained CLARITY-cleared brains, however efficiently labelled uDISCO and ECi-cleared brains. Conversely, and in contrast to ORO-staining, DiI equally stained control, CLARITY, ECi and uDISCO-cleared brains. Both ORO- and DiI-staining demonstrated impairment in brain tissue integrity following CLARITY, but less so in uDISCO and ECi brains. DiI-washout assay demonstrated that each of the solvents employed along the process of uDISCO and ECi are highly delipidating, as well as the SDS-electrophoresis employed during CLARITY clearing. However, Scale treatment preserved most of the DiI dye. These data emphasize the variability in lipid assessment of cleared tissues by common techniques, and may help to resolve the contribution of lipids in brain MRI contrast.

Design of Ultrapotent Genetically Encoded Inhibitors of Kv4.2 for Gating Neural Plasticity.

The Kv4.2 potassium channel plays established roles in neuronal excitability, while also being implicated in plasticity. Current means to study the roles of Kv4.2 are limited, motivating us to design a genetically encoded membrane tethered Heteropodatoxin-2 (MetaPoda). We find that MetaPoda is an ultrapotent and selective gating-modifier of Kv4.2. We narrow its site of contact with the channel to two adjacent residues within the voltage sensitive domain (VSD) and, with docking simulations, suggest that the toxin binds the VSD from within the membrane. We also show that MetaPoda does not require an external linker of the channel for its activity. In neurons (obtained from female and male rat neonates), MetaPoda specifically, and potently, inhibits all Kv4 currents, leaving all other A-type currents unaffected. Inhibition of Kv4 in hippocampal neurons does not promote excessive excitability, as is expected from a simple potassium channel blocker. We do find that MetaPoda's prolonged expression (1 week) increases expression levels of the immediate early gene cFos and prevents potentiation. These findings argue for a major role of Kv4.2 in facilitating plasticity of hippocampal neurons. Lastly, we show that our engineering strategy is suitable for the swift engineering of another potent Kv4.2-selective membrane-tethered toxin, Phrixotoxin-1, denoted MetaPhix. Together, we provide two uniquely potent genetic tools to study Kv4.2 in neuronal excitability and plasticity.

A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo.

The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to transduction by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is markedly lower than in rodents; likely holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing, patch clamp electrophysiology and calcium imaging. We then leveraged the cultures for the rapid screening of various viruses, only to find that all yielded poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quails led us to rationally design a custom-made AAV variant (AAV1-T593K; AAV1*) that exhibits improved transduction efficiencies in vitro and in vivo (14- and five-fold, respectively). Together, we present unique culturing method, transcriptomic profiles of quail's brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.

Differential roles for DNAJ isoforms in HTT-polyQ and FUS aggregation modulation revealed by chaperone screens.

Protein aggregation is a hallmark of neurodegeneration. Here, we find that Huntington's disease-related HTT-polyQ aggregation induces a cellular proteotoxic stress response, while ALS-related mutant FUS (mutFUS) aggregation leads to deteriorated proteostasis. Further exploring chaperone function as potential modifiers of pathological aggregation in these contexts, we reveal divergent effects of naturally-occurring chaperone isoforms on different aggregate types. We identify a complex of the full-length (FL) DNAJB14 and DNAJB12, that substantially protects from mutFUS aggregation, in an HSP70-dependent manner. Their naturally-occurring short isoforms, however, do not form a complex, and lose their ability to preclude mutFUS aggregation. In contrast, DNAJB12-short alleviates, while DNAJB12-FL aggravates, HTT-polyQ aggregation. DNAJB14-FL expression increases the mobility of mutFUS aggregates, and restores the deteriorated proteostasis in mutFUS aggregate-containing cells and primary neurons. Our results highlight a maladaptive cellular response to pathological aggregation, and reveal a layer of chaperone network complexity conferred by DNAJ isoforms, in regulation of different aggregate types.

Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy.

The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding, which we confirm by functional analysis. Both variants show three orders of magnitude reductions in glutamate EC50, with G689S exhibiting the largest reductions observed for GRIN2B (~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2Bwt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the variants fail to respond due to G689's novel role in proton-sensing. Together, we describe two unique variants with extreme effects on channel function. We employ protein-stability measures to explain why current (and future) LBD mutations in GluN2B primarily instigate Loss-of-Function.

Photo-transformable genetically-encoded optical probes for functional highlighting in vivo.

Studying the brain requires knowledge about both structure (i.e., connectome) and function of its constituents (neurons and glia alike). This need has prompted the development of novel tools and techniques, in particular optical techniques to examine cells remotely. Early works (1900's) led to the development of novel cell-staining techniques that, when combined with the use of a very simple light microscope, visualized individual neurons and their subcellular compartments in fixed tissues. Today, highlighting of structure and function can be performed on live cells, notably in vivo, owing to discovery of GFP and subsequent development of genetically encoded fluorescent optical tools. In this review, we focus our attention on a subset of optical biosensors, namely probes whose emission can be modified by light. We designate them photo-transformable genetically encoded probes. The family of photo-transformable probes embraces current probes that undergo photoactivation (PA), photoconversion (PC) or photoswitching (PS). We argue that these are particularly suited for studying multiple features of neurons, such as structure, connectivity and function concomitantly, for functional highlighting of neurons in vivo.

ATF3 Regulates the Expression of AChE During Stress.

Acetylcholinesterase (AChE) expresses in non-cholinergic cells, but its role(s) there remain unknown. We have previously attributed a pro-apoptotic role for AChE in stressed retinal photoreceptors, though by unknown mechanism. Here, we examined its promoter only to find that it includes a binding sequence for the activating transcription factor 3 (ATF3); a prototypical mediator of apoptosis. This suggests that expression of AChE could be regulated by ATF3 in the retina. Indeed, ATF3 binds the AChE-promoter to down-regulate its expressions in vitro. Strikingly, retinas of "blinded" mice display hallmarks of apoptosis, almost exclusively in the outer nuclear layer (ONL); coinciding with elevated levels of AChE and absence of ATF3. A mirror image is observed in the inner nuclear layer (INL), namely prominent levels of ATF3 and lack of AChE as well as lack of apoptosis. We conclude that segregated patterns of expressions of ATF3 reflect its ability to repress apoptosis in different layers of the retina-a novel mechanism behind apoptosis.

The ubiquitously expressed bZIP inhibitor, JDP2, suppresses the transcription of its homologue immediate early gene counterpart, ATF3.

JDP2 is a ubiquitously expressed bZIP repressor protein. JDP2 binds TPA response element and cyclic AMP response element located within various promoters. JDP2 displays a high degree of homology to the immediate early gene ATF3. ATF3 plays a crucial role in the cellular adaptive response to multiple stress insults as well as growth stimuli. We have identified ATF3 as a potential target gene for JDP2 repression. JDP2 regulates the ATF3 promoter potentially through binding to both the consensus ATF/CRE site and a non-consensus ATF3 auto-repression DNA-binding element. Expression of ATF3 protein in wild-type mouse embryo fibroblast (MEF) cells is below the detectable levels, whereas, JDP2 disrupted MEF cells display noticeable level of ATF3 protein. Following either serum or ER stress stimulation, ATF3 expression is potentiated in JDP2-KO fibroblast cells as compared with wild-type cells. Mice with either JDP2 over-expression or JDP2 disruption display undetectable level of ATF3 protein. However, ATF3 induction in response to either growth or stress signals is dependent on JDP2 expression level. ATF3 induction is attenuated in JDP2 over-expressing mice whereas is potentiated in JDP2-KO mice as compared with the corresponding wild-type mice. Collectively, the data presented strongly suggest that JDP2 plays a role in the determination of the ATF3 adaptive cellular threshold response to different stress insults and growth stimuli.

Inhibition of basic leucine zipper transcription is a major mediator of atrial dilatation.

OBJECTIVE: Atrial fibrillation is the most prevalent clinically significant cardiac arrhythmia. Atrial dilatation, a predictor of atrial fibrillation, is thought to result from increased ventricular pressure. However, the underlying molecular mechanisms responsible for atrial dilatation are largely unknown. Here we sought to examine whether the expression of a basic leucine zipper inhibitor protein, JDP2, in the heart is sufficient for the generation of atrial dilatation. METHODS: A tetracycline-regulated transgene was used to express JDP2 specifically in the mouse heart. Mice hearts were dissected and subjected to Northern and Western analysis, or analyzed by ECG recording and echocardiography. Regulation of gene expression was studied using electromobility shift assays and luciferase gene reporter analysis. RESULTS: Expression of JDP2 resulted in massive bi-atrial dilatation, defects in conduction, and a lethal phenotype. These effects were developmentally independent, acquired during adulthood, and were reversible upon abolishing of JDP2 expression. Connexin 40 and myosin light chain 2a expression were identified as potential target genes. CONCLUSION: Expression of basic leucine zipper transcription inhibitors is sufficient to results in atrial dilatation. This dilatation is acquired postnatally and is reversible. Thus, basic leucine zipper transcription inhibitors may be a relevant therapeutic target for preventing atrial dilatation and atrial fibrillation.

The c-Jun dimerization protein 2 inhibits cell transformation and acts as a tumor suppressor gene.

The c-Jun dimerization protein, JDP2, is a member of the AP-1 (activating protein-1) family of the basic leucine zipper transcription factors. JDP2 can bind 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and cAMP-responsive element DNA response elements, resulting in the inhibition of transcription. Although the role of AP-1 in cell proliferation and malignant transformation is well established, the role of JDP2 in this process is of subject to debate. On the one hand, JDP2 was shown to inhibit cyclin D transcription and promote differentiation of skeletal muscle and osteoclast cells. On the other hand, JDP2 was shown to partially transform chicken embryo fibroblast and was identified in a screen for oncogenes able to collaborate with the loss of p27kip cyclin-dependent inhibitor to induce lymphomas. Using cell transformation assays in NIH3T3 cells and injection of prostate cancer cell lines overexpressing JDP2 into severe combined immuno-deficient (SCID) mice, we show for the first time the potential role of JDP2 in inhibition of cell transformation and tumor suppression. The mechanism of tumor suppressor action of JDP2 can be partially explained by the generation of inhibitory AP-1 complexes via the increase of JunB, JunD, and Fra2 expression and decrease of c-Jun expression.

Oxidative stress increases the expression of the angiotensin-II receptor type 1 in mouse peritoneal macrophages.

Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type 1 (AT(1))-receptor mediates most of Ang II-induced pro-atherogenic effects. In this study we have examined the effect of macrophage oxidative stress on cellular AT(1)-receptor expression. Mouse peritoneal macrophages (MPM) from apolipoprotein-E deficient (E(0)) mice at increasing ages (1 6 months) demonstrated an age-dependent increase in cellular lipid-peroxides (PD) content. In parallel, the AT(1)-receptor mRNA and protein levels both increased by up to 3.7-fold and 1.7-fold, respectively, in MPM from 6-month old mice compared with 1-month old mice. Vitamin E supplementation to E(0) mice significantly decreased the MPM PD content and macrophage AT(1)-receptor mRNA expression compared with placebo-treated mice. The role of oxidative stress in the cellular expression of AT(1)-receptors was further demonstrated by manipulation of macrophage glutathione content. Buthionine-sulfoximine, a glutathione synthesis inhibitor, increased MPM PD content and AT(1)-receptor mRNA expression, whereas L-2-oxothiazolidine-4-carboxylic acid, that contributes to glutathione synthesis, reduced macrophage PD and AT(1)-receptor mRNA expression. Incubation of MPM with oxidised low-density lipoproteins (LDL) led to a significant, dose-dependent and time-dependent increase in macrophage AT(1)-receptor mRNA and protein expression, compared with control cells. In contrast, native LDL or acetylated LDL did not significantly affect macrophage AT(1)-receptor mRNA expression. In conclusion, our findings suggest that oxidative stress in macrophages induces AT(1)-receptor expression. This phenomenon can stimulate the interaction of Ang II with macrophages and hence accelerate macrophage foam cell formation and early atherogenesis.