Fluri F, Malzahn U, Homola GA, Schuhmann MK, Kleinschnitz C, Volkmann J.
PMID: 29059697 | DOI: 10.1002/ana.25086
Abstract
OBJECTIVE:
One-third of all stroke survivors are unable to walk, even after intensive physiotherapy. Thus, other concepts to restore walking are needed. Since electrical stimulation of the mesencephalic locomotor region (MLR) is known to elicit gait movements, this area might be a promising target for restorative neurostimulation in stroke patients with gait disability. The present study aims to delineate the effect of high-frequency stimulation of the MLR (MLR-HFS) on gait impairment in a rodent stroke model.
METHODS:
Male Wistar rats underwent photothrombotic stroke of the right sensorimotor cortex and chronic implantation of a stimulating electrode into the right MLR. Gait was assessed using clinical scoring of the beam walking test and videokinematic analysis (CatWalk™) at baseline and on days 3 and 4 after experimental stroke with and without MLR-HFS.
RESULTS:
Kinematic analysis revealed significant changes in several dynamic and static gait parameters resulting in overall reduced gait velocity. All rats exhibited major coordination deficits during the beam walking challenge and were unable to cross the beam. Simultaneous to the onset of MLR-HFS, a significantly higher walking speed and improvements in several dynamic gait parameters were detected by the Catwalk™-system. Rats regained the ability to cross the beam unassisted showing a reduced number of paw slips and misses.
INTERPRETATION:
MLR-HFS can improve disordered locomotor function in a rodent stroke model. It may act by shielding brainstem and spinal locomotor centers from abnormal cortical input after stroke, thus allowing for compensatory and independent action of these circuits.
Biochemical and biophysical research communications
Lin, C;Xiao, Z;Zhang, X;Wu, G;
PMID: 35430449 | DOI: 10.1016/j.bbrc.2022.04.034
Circular RNAs (circRNAs) are a class of noncoding RNAs generated by a specific type of RNA alternative splicing called backsplicing through various mechanisms. Recently, thousands of circRNAs have been identified by high-throughput RNA sequencing technologies and bioinformatics analysis. However, the functions of the majority have not been fully elucidated yet. Different tools, such as in situ hybridization, can help visualize the spatial temporal distribution of circRNA molecules, thus assisting the understanding of their biological and physiological functions. Here, we present a simple and straightforward method based on padlock probe hybridization and rolling circle amplification (RCA) for in situ detection of circRNAs. We compared our method with the commercially available BaseScope assay for the detection of Cdr1as in the mouse brain tissue. The result showed that the two methods have achieved comparable detection efficiency, thus demonstrating our padlock probe assay as an alternative yet simple circRNA in situ detection method for the research community.
AAV9-mediated FIG4 delivery prolongs life span in Charcot Marie Tooth disease type 4J mouse model
The Journal of clinical investigation
Presa, M;Bailey, RM;Davis, C;Murphy, T;Cook, J;Walls, R;Wilpan, H;Bogdanik, L;Lenk, GM;Burgess, RW;Gray, SJ;Lutz, C;
PMID: 33878035 | DOI: 10.1172/JCI137159
Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten lifespan. There is currently no treatment for CMT4J. Here we present the results of preclinical studies testing a gene therapy approach to restore FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded AAV9 to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at postnatal day 1 or 4, mice survived at least one year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When treated at postnatal day 7 or 11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.
Vieira, JR;Shah, B;Dupraz, S;Paredes, I;Himmels, P;Schermann, G;Adler, H;Motta, A;Gärtner, L;Navarro-Aragall, A;Ioannou, E;Dyukova, E;Bonnavion, R;Fischer, A;Bonanomi, D;Bradke, F;Ruhrberg, C;Ruiz de Almodóvar, C;
PMID: 36549270 | DOI: 10.1016/j.neuron.2022.12.005
How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.
Ali Marandi Ghoddousi, R;Magalong, VM;Kamitakahara, AK;Levitt, P;
PMID: 36313803 | DOI: 10.1016/j.crmeth.2022.100316
Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.
Hong, DS;Van Tine, BA;Biswas, S;McAlpine, C;Johnson, ML;Olszanski, AJ;Clarke, JM;Araujo, D;Blumenschein, GR;Kebriaei, P;Lin, Q;Tipping, AJ;Sanderson, JP;Wang, R;Trivedi, T;Annareddy, T;Bai, J;Rafail, S;Sun, A;Fernandes, L;Navenot, JM;Bushman, FD;Everett, JK;Karadeniz, D;Broad, R;Isabelle, M;Naidoo, R;Bath, N;Betts, G;Wolchinsky, Z;Batrakou, DG;Van Winkle, E;Elefant, E;Ghobadi, A;Cashen, A;Grand'Maison, A;McCarthy, P;Fracasso, PM;Norry, E;Williams, D;Druta, M;Liebner, DA;Odunsi, K;Butler, MO;
PMID: 36624315 | DOI: 10.1038/s41591-022-02128-z
Affinity-optimized T cell receptors can enhance the potency of adoptive T cell therapy. Afamitresgene autoleucel (afami-cel) is a human leukocyte antigen-restricted autologous T cell therapy targeting melanoma-associated antigen A4 (MAGE-A4), a cancer/testis antigen expressed at varying levels in multiple solid tumors. We conducted a multicenter, dose-escalation, phase 1 trial in patients with relapsed/refractory metastatic solid tumors expressing MAGE-A4, including synovial sarcoma (SS), ovarian cancer and head and neck cancer ( NCT03132922 ). The primary endpoint was safety, and the secondary efficacy endpoints included overall response rate (ORR) and duration of response. All patients (N = 38, nine tumor types) experienced Grade ≥3 hematologic toxicities; 55% of patients (90% Grade ≤2) experienced cytokine release syndrome. ORR (all partial response) was 24% (9/38), 7/16 (44%) for SS and 2/22 (9%) for all other cancers. Median duration of response was 25.6 weeks (95% confidence interval (CI): 12.286, not reached) and 28.1 weeks (95% CI: 12.286, not reached) overall and for SS, respectively. Exploratory analyses showed that afami-cel infiltrates tumors, has an interferon-γ-driven mechanism of action and triggers adaptive immune responses. In addition, afami-cel has an acceptable benefit-risk profile, with early and durable responses, especially in patients with metastatic SS. Although the small trial size limits conclusions that can be drawn, the results warrant further testing in larger studies.
Kathe, C;Skinnider, MA;Hutson, TH;Regazzi, N;Gautier, M;Demesmaeker, R;Komi, S;Ceto, S;James, ND;Cho, N;Baud, L;Galan, K;Matson, KJE;Rowald, A;Kim, K;Wang, R;Minassian, K;Prior, JO;Asboth, L;Barraud, Q;Lacour, SP;Levine, AJ;Wagner, F;Bloch, J;Squair, JW;Courtine, G;
PMID: 36352232 | DOI: 10.1038/s41586-022-05385-7
A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord<sup>1-3</sup> applied during neurorehabilitation<sup>4,5</sup> (EES<sup>REHAB</sup>) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential for a patient to walk after spinal cord injury. To identify these putative neurons, we modelled the technological and therapeutic features underlying EES<sup>REHAB</sup> in mice. We applied single-nucleus RNA sequencing<sup>6-9</sup> and spatial transcriptomics<sup>10,11</sup> to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type<sup>12,13</sup> and spatial prioritization to identify the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons are not required for walking before spinal cord injury, we demonstrate that they are essential for the recovery of walking with EES following spinal cord injury. Augmenting the activity of these neurons phenocopied the recovery of walking enabled by EES<sup>REHAB</sup>, whereas ablating them prevented the recovery of walking that occurs spontaneously after moderate spinal cord injury. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after paralysis. Moreover, our methodology establishes a framework for using molecular cartography to identify the neurons that produce complex behaviours.
Greguske, EA;Maroto, AF;Borrajo, M;Palou, A;Gut, M;Esteve-Codina, A;Barrallo-Gimeno, A;Llorens, J;
PMID: 37100209 | DOI: 10.1016/j.nbd.2023.106134
The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.
Skirzewski, M;Princz-Lebel, O;German-Castelan, L;Crooks, AM;Kim, GK;Tarnow, SH;Reichelt, A;Memar, S;Palmer, D;Li, Y;Jane Rylett, R;Saksida, LM;Prado, VF;Prado, MAM;Bussey, TJ;
PMID: 36564387 | DOI: 10.1038/s41467-022-35601-x
The ability to learn Pavlovian associations from environmental cues predicting positive outcomes is critical for survival, motivating adaptive behaviours. This cued-motivated behaviour depends on the nucleus accumbens (NAc). NAc output activity mediated by spiny projecting neurons (SPNs) is regulated by dopamine, but also by cholinergic interneurons (CINs), which can release acetylcholine and glutamate via the activity of the vesicular acetylcholine transporter (VAChT) or the vesicular glutamate transporter (VGLUT3), respectively. Here we investigated behavioural and neurochemical changes in mice performing a touchscreen Pavlovian approach task by recording dopamine, acetylcholine, and calcium dynamics from D1- and D2-SPNs using fibre photometry in control, VAChT or VGLUT3 mutant mice to understand how these signals cooperate in the service of approach behaviours toward reward-predicting cues. We reveal that NAc acetylcholine-dopaminergic signalling is continuously updated to regulate striatal output underlying the acquisition of Pavlovian approach learning toward reward-predicting cues.
Liau, ES;Jin, S;Chen, YC;Liu, WS;Calon, M;Nedelec, S;Nie, Q;Chen, JA;
PMID: 36596814 | DOI: 10.1038/s41467-022-35574-x
Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.
Coverdell, TC;Abraham-Fan, RJ;Wu, C;Abbott, SBG;Campbell, JN;
PMID: 35705034 | DOI: 10.1016/j.celrep.2022.110962
Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus that also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2+ fibers in the esophagus results in contractions. Activating Crhr2nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all of the nAmb neurons robustly suppresses heart rate. Together, these results reveal a genetically defined circuit for motor control of the esophagus.
Cho, I;Chang, JB;
PMID: 35233025 | DOI: 10.1038/s41598-022-06903-3
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.
