Biology subjects

Le Pichon, C. E.

Publications and source records attributed to Le Pichon, C. E..

5 recordsLinked to original sources

bioRxiv2024

The transcriptional response of cortical neurons to concussion reveals divergent fates after injury

Traumatic brain injury (TBI) is a risk factor for neurodegeneration, however little is known about how different neuron types respond to this kind of injury. In this study, we follow neuronal populations over several months after a single mild TBI (mTBI) to assess long ranging consequences of injury at the level of single, transcriptionally defined neuronal classes. We find that the stress responsive Activating Transcription Factor 3 (ATF3) defines a population of cortical neurons after mTBI. We show that neurons that activate ATF3 upregulate stress-related genes while repressing many genes, including commonly used markers for these cell types. Using an inducible reporter linked to ATF3, we genetically mark damaged cells to track them over time. Notably, we find that a population in layer V undergoes cell death acutely after injury, while another in layer II/III survives long term and retains the ability to fire action potentials. To investigate the mechanism controlling layer V neuron death, we genetically silenced candidate stress response pathways. We found that the axon injury responsive kinase MAP3K12, also known as dual leucine zipper kinase (DLK), is required for the layer V neuron death. This work provides a rationale for targeting the DLK signaling pathway as a therapeutic intervention for traumatic brain injury. Beyond this, our novel approach to track neurons after a mild, subclinical injury can inform our understanding of neuronal susceptibility to repeated impacts.

neuroscience↗

bioRxiv2023

Creation of de novo cryptic splicing for ALS/FTD precision medicine

A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe "TDP-REG", a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF in vitro and in vivo, including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the UNC13A cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals. One-Sentence SummaryWe engineer TDP-43-regulated cryptic exons, enabling exceptionally precise activation of gene therapies in diseased neurons.

molecular biology↗

bioRxiv2023

ATF2 phosphorylation is a key event in neuronal apoptosis, linking the DLK/LZK kinase cascade to JUN upregulation

Apoptotic neuron death is a key feature of neurodegenerative disease. Considerable efforts have been made to target this pathway but the molecular mechanisms remain incompletely understood. Here, we conducted an unbiased whole genome CRISPR inhibition screen in human neurons to discover genes required for their death and identified known targets including the kinase MAP3K12 (DLK) and the transcription factor JUN. In addition, this screen revealed a potential role for the transcription factor ATF2. We demonstrate that ATF2 phosphorylation by MAP3 kinases is the core driver of the pro-apoptotic transcriptional response. Surprisingly, JUN phosphorylation is not required for apoptosis. However, the phosphorylation of ATF2 and upregulation of JUN expression are crucial. ATF2 therefore converts the kinase signal into a transcriptional response. Inhibiting ATF2 in cultured human neurons prevents cell death. Notably we show that ATF2 knockdown is neuroprotective in injury models in vivo. Thus, ATF2 provides a promising new target for a wide range of neurodegenerative disorders.

neuroscience↗

bioRxiv2023

DLK-dependent mitochondrial fission drives axon degeneration and neuronal cell death

Currently there are no effective treatments for an array of neurodegenerative disorders to a large part because cell-based models fail to recapitulate disease. Here we developed a reproducible human iPSC-based model where laser axotomy causes retrograde axon degeneration leading to neuronal cell death. Time-lapse confocal imaging revealed that damage triggers an apoptotic wave of mitochondrial fission proceeding from the site of injury to the soma. We demonstrated that this apoptotic wave is locally initiated in the axon by dual leucine zipper kinase (DLK). We found that mitochondrial fission and resultant cell death are entirely dependent on phosphorylation of dynamin related protein 1 (DRP1) downstream of DLK, revealing a new mechanism by which DLK can drive apoptosis. Importantly, we show that CRISPR mediated Drp1 depletion protected mouse retinal ganglion neurons from degeneration after optic nerve crush. Our results provide a powerful platform for studying degeneration of human neurons, pinpoint key early events in damage related neural death and new focus for therapeutic intervention.

neuroscience↗

bioRxiv2020

Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we developed a targeted single nuclear RNA sequencing approach and used it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their extremely rich diversity. Specifically, visceral motor neurons, which provide autonomic control, could be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons was also reflected in our analysis with alpha, beta, and gamma subtypes clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

neuroscience↗