Biology subjects

Boutin, J.

Publications and source records attributed to Boutin, J..

7 recordsLinked to original sources

bioRxiv2025

Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells

Genome editing by CRISPR-Cas9 is promising for gene therapy. However, safety concerns remain, particularly regarding the ON-target genotoxicity associated with protocols using nucleases. Monitoring the genotoxicity of edited cells before and after graft is essential, especially to assay potentially deleterious megabase-scale genomic rearrangements induced at the targeted locus. High sensitivity requires single-cell resolution. Here, we developed an integrated approach combining targeted single-cell DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq) with complementary micronuclei and LOH cytometry-reporter assays. This multiplexed strategy enables orthogonal readouts to accurately monitor CRISPR-mediated genotoxicity in primary cells. Using this approach, we detected, mapped and characterized various types of induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). Our compelling workflow assessed editing-associated chromosomal instability linked to double strand break after editing. Importantly, palbociclib prevented the appearance of such genomic rearrangements in hematopoietic stem/progenitor cells without impairing cell fate or graft capability. Conversely, short-term risk was significantly increased with DNA-PKcs inhibitor AZD7648 (HDR booster) in HSPCs and fibroblasts. Fortunately, targeting HBG1/2 in Chr11p in HSPCs, scSNP-DNA-seq revealed that ON-target genotoxic events were no longer detectable after long-term xenografts, even in AZD7648-treated cells. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.

bioengineering↗

bioRxiv2025

Cell-type specific sensory and motor activity in the cuneiform nucleus and pedunculopontine nucleus in mice

The activity of neurotransmitter-based cell types in the cuneiform and pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation is not fully understood. Using fiber photometry in mice, we found cell-type specific responses to sensory stimuli. Glutamatergic and GABAergic cells responded to sound, visual looming, and air puffs, except for pedunculopontine GABAergic cells, which did not respond to visual looming. Cholinergic cells responded to air puffs. When a stimulus triggered high-speed locomotion, activity increased in cuneiform glutamatergic neurons. Conversely, when low-speed locomotion was triggered, activity increased in pedunculopontine glutamatergic neurons. During spontaneous low-speed locomotion, activity increased in pedunculopontine glutamatergic cells. Activity also increased in a cell type-specific manner during grooming or rearing. Our study shows cell type-specific activity in the cuneiform or pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation. Sensory responsiveness likely has relevance in Parkinsons disease, where sensory circuits are increasingly targeted to improve walking.

neuroscience↗

bioRxiv2025

ZIP editors for efficient and versatile CRISPR-Cas9 precise genome editing

Precise genome editing technologies create the potential for genetic studies and innovative gene therapies. Here we present new CRISPR-Cas9 tools, named ZIP CRISPR, loaded with a single-stranded oligodeoxynucleotide (ssODN) template on the Cas ribonucleoprotein complex. The ssODN template is annealed to an extended guide RNA (gRNA) allowing its nuclear delivery at the right place, i.e. the targeted DNA cut, and at the right time. This new template import system is easy-to-design, easy-to-use, inexpensive and versatile. It increases homology-directed repair (HDR) editing efficiency using Cas9 nuclease up to 12-fold with a mean increase of 5-fold as demonstrated at many loci in many cell types. Based on a heteroduplex gRNA-ssODN, it can also be used with the Cas9 nickase, resulting in HDR editing with minimal InDels, and preventing double-strand break (DSB)-mediated genotoxicity. ZIP CRISPR is a non-viral platform adaptable to targeted DSB (higher HDR editing efficiency) or nick (higher safety) to precisely model and correct a wide range of edits. It is suitable for many biological applications and could be considered for HDR-based gene therapies.

bioengineering↗

bioRxiv2024

A cortical locus for modulation of arousal states

Fluctuations in global arousal are key determinants of spontaneous cortical activity and function. Several subcortical structures, including neuromodulatory nuclei like the locus coeruleus (LC), are involved in the regulation of arousal. However, much less is known about the role of cortical circuits that provide top-down inputs to arousal-related subcortical structures. Here, we investigated the role of a major subdivision of the prefrontal cortex, the anterior cingulate cortex (ACC), in arousal modulation. Pupil size, facial movements, heart rate, and locomotion were used as non-invasive measures of arousal and behavioral state. We designed a closed loop optogenetic system based on machine vision and found that real time inhibition of ACC activity during pupil dilations suppresses ongoing arousal events. In contrast, inhibiting activity in a control cortical region had no effect on arousal. Fiber photometry recordings showed that ACC activity scales with the magnitude of spontaneously occurring pupil dilations/face movements independently of locomotion. Moreover, optogenetic ACC activation increases arousal independently of locomotion. In addition to modulating global arousal, ACC responses to salient sensory stimuli scaled with the size of evoked pupil dilations. Consistent with a role in sustaining saliency-linked arousal events, pupil responses to sensory stimuli were suppressed with ACC inactivation. Finally, our results comparing arousal-related ACC and norepinephrinergic LC neuron activity support a role for the LC in initiation of arousal events which are modulated in real time by the ACC. Collectively, our experiments identify the ACC as a key cortical site for sustaining momentary increases in arousal and provide the foundation for understanding cortical-subcortical dynamics underlying the modulation of arousal states.

neuroscience↗

bioRxiv2022

Apelin Stimulation of the Perivascular MuSC Niche Enhances Endogenous Repair in Muscular Dystrophy

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here we describe that defects in the endothelial cell (EC) compartment of the perivascular stem cell niche in three different types of MD are associated with inefficient mobilization of MuSCs following tissue damage. Using chemoinformatic analysis, we identified the 13 amino acid form of the peptidic hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. In dystrophic mice, administration of AP-13 generates a pro-myogenic EC-rich niche that supports MuSC function and markedly improves tissue regeneration, muscle strength, and physical performance. Moreover, we demonstrate that EC specific knockout of the AP-13 receptor leads to regenerative defects that phenocopy major pathological features of MD. Altogether, we provide in vivo proof-of-concept that enhancing endogenous repair by targeting the perivascular niche is a viable therapeutic avenue for MD and characterize AP-13 as a novel drug candidate for systemic treatment of stem cell dysfunction.

cell biology↗

bioRxiv2021

Optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus controls locomotor movements in a mouse model of Parkinson's disease

In Parkinsons disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits such a gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to decreased activity in the Mesencephalic Locomotor Region (MLR), a brainstem region controlling locomotion. Clinicians are exploring deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of Parkinsons disease based on bilateral striatal injection of 6-hydroxydopamine (6-OHDA), which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in freely moving animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open new avenues to develop targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy or optogenetics. SIGNIFICANCE STATEMENTIn Parkinsons disease, alleviating locomotor deficits is a challenge. Clinicians are exploring deep brain stimulation of the Mesencephalic Locomotor Region, a brainstem region controlling locomotion, but results are mixed. However, the best target in this region in Parkinsons disease remains unknown. Indeed, this region which comprises the pedunculopontine and cuneiform nuclei, contains different cell types with opposing effects on locomotor output. Here, using a mouse model where midbrain dopaminergic cells were damaged by a neurotoxin, we demonstrate that optogenetic activation of glutamatergic neurons in the cuneiform nucleus increases locomotion, controls speed, and evokes limb movements similar to those observed during spontaneous locomotion in intact animals. Our study identifies a potentially clinically relevant target to improve locomotor function in Parkinsons disease.

neuroscience↗

bioRxiv2020

Freely behaving mice can brake and turn during optogenetic stimulation of the Mesencephalic Locomotor Region

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSStimulation of the Mesencephalic Locomotor Region (MLR) is increasingly considered as a target to improve locomotor function in Parkinsons disease, spinal cord injury and stroke. A key function of the MLR is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Objective/hypothesisWe investigated whether freely behaving mice could brake or turn based on environmental cues during MLR stimulation. MethodsWe stimulated the cuneiform nucleus in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. ResultsOptogenetic stimulation of the MLR evoked locomotion and increasing laser power increased locomotor speed. Gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. Mice could brake and make sharp turns ([~]90) when approaching a corner during MLR stimulation in an open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turn angle. In a reporter mouse, many Vglut2-ZsGreen neurons were immunopositive for glutamate in the MLR. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short latency spiking in MLR neurons. ConclusionMLR glutamatergic neurons are a relevant target to improve locomotor activity without impeding the ability to brake and turn when approaching an obstacle, thus ensuring smooth and adaptable navigation. HO_SCPLOWIGHLIGHTSC_SCPLOW- Mice brake and turn when approaching the arenas corner during MLR-evoked locomotion - Speed decrease is scaled to speed before the turn during MLR-evoked locomotion - Turn angle is scaled to turn speed during MLR-evoked locomotion - Gait and limb kinematics are similar during spontaneous and MLR-evoked locomotion

neuroscience↗