Institut des Neurosciences Paris-Saclay

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

BACKGROUND: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. RESULTS: We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. CONCLUSIONS: To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

INTRODUCTION The cerebral cortex underlies our complex cognitive capabilities. Variations in human cortical surface area and thickness are associated with neurological, psychological, and behavioral traits and can be measured in vivo by magnetic resonance imaging (MRI). Studies in model organisms have identified genes that influence cortical structure, but little is known about common genetic variants that affect human cortical structure. RATIONALE To identify genetic variants associated with human cortical structure at both global and regional levels, we conducted a genome-wide association meta-analysis of brain MRI data from 51,665 individuals across 60 cohorts. We analyzed the surface area and average thickness of the whole cortex and 34 cortical regions with known functional specializations. RESULTS We identified 369 nominally genome-wide significant loci ( P < 5 × 10 −8 ) associated with cortical structure in a discovery sample of 33,992 participants of European ancestry. Of the 360 loci for which replication data were available, 241 loci influencing surface area and 66 influencing thickness remained significant after replication, with 237 loci passing multiple testing correction ( P < 8.3 × 10 −10 ; 187 influencing surface area and 50 influencing thickness). Common genetic variants explained 34% (SE = 3%) of the variation in total surface area and 26% (SE = 2%) in average thickness; surface area and thickness showed a negative genetic correlation ( r G = −0.32, SE = 0.05, P = 6.5 × 10 −12 ), which suggests that genetic influences have opposing effects on surface area and thickness. Bioinformatic analyses showed that total surface area is influenced by genetic variants that alter gene regulatory activity in neural progenitor cells during fetal development. By contrast, average thickness is influenced by active regulatory elements in adult brain samples, which may reflect processes that occur after mid-fetal development, such as myelination, branching, or pruning. When considered together, these results support the radial unit hypothesis that different developmental mechanisms promote surface area expansion and increases in thickness. To identify specific genetic influences on individual cortical regions, we controlled for global measures (total surface area or average thickness) in the regional analyses. After multiple testing correction, we identified 175 loci that influence regional surface area and 46 that influence regional thickness. Loci that affect regional surface area cluster near genes involved in the Wnt signaling pathway, which is known to influence areal identity. We observed significant positive genetic correlations and evidence of bidirectional causation of total surface area with both general cognitive functioning and educational attainment. We found additional positive genetic correlations between total surface area and Parkinson’s disease but did not find evidence of causation. Negative genetic correlations were evident between total surface area and insomnia, attention deficit hyperactivity disorder, depressive symptoms, major depressive disorder, and neuroticism. CONCLUSION This large-scale collaborative work enhances our understanding of the genetic architecture of the human cerebral cortex and its regional patterning. The highly polygenic architecture of the cortex suggests that distinct genes are involved in the development of specific cortical areas. Moreover, we find evidence that brain structure is a key phenotype along the causal pathway that leads from genetic variation to differences in general cognitive function. Identifying genetic influences on human cortical structure. ( A ) Measurement of cortical surface area and thickness from MRI. ( B ) Genomic locations of common genetic variants that influence global and regional cortical structure. ( C ) Our results support the radial unit hypothesis that the expansion of cortical surface area is driven by proliferating neural progenitor cells. ( D ) Cortical surface area shows genetic correlation with psychiatric and cognitive traits. Error bars indicate SE. IMAGE CREDITS: (A) K. COURTNEY; (C) M. R. GLASS

Balance of excitation and inhibition is a fundamental feature of in vivo network activity and is important for its computations. However, its presence in the neocortex of higher mammals is not well established. We investigated the dynamics of excitation and inhibition using dense multielectrode recordings in humans and monkeys. We found that in all states of the wake-sleep cycle, excitatory and inhibitory ensembles are well balanced, and co-fluctuate with slight instantaneous deviations from perfect balance, mostly in slow-wave sleep. Remarkably, these correlated fluctuations are seen for many different temporal scales. The similarity of these computational features with a network model of self-generated balanced states suggests that such balanced activity is essentially generated by recurrent activity in the local network and is not due to external inputs. Finally, we find that this balance breaks down during seizures, where the temporal correlation of excitatory and inhibitory populations is disrupted. These results show that balanced activity is a feature of normal brain activity, and break down of the balance could be an important factor to define pathological states.

A given cell makes exchanges with its neighbors through a variety of means ranging from diffusible factors to vesicles. Cells use also tunneling nanotubes (TNTs), filamentous-actin-containing membranous structures that bridge and connect cells. First described in immune cells, TNTs facilitate HIV-1 transfer and are found in various cell types, including neurons. We show that the microtubule-associated protein Tau, a key player in Alzheimer's disease, is a bona fide constituent of TNTs. This is important because Tau appears beside filamentous actin and myosin 10 as a specific marker of these fine protrusions of membranes and cytosol that are difficult to visualize. Furthermore, we observed that exogenous Tau species increase the number of TNTs established between primary neurons, thereby facilitating the intercellular transfer of Tau fibrils. In conclusion, Tau may contribute to the formation and function of the highly dynamic TNTs that may be involved in the prion-like propagation of Tau assemblies.

Extracting neuronal spiking activity from large-scale two-photon recordings remains challenging, especially in mammals in vivo, where large noises often contaminate the signals. We propose a method, MLspike, which returns the most likely spike train underlying the measured calcium fluorescence. It relies on a physiological model including baseline fluctuations and distinct nonlinearities for synthetic and genetically encoded indicators. Model parameters can be either provided by the user or estimated from the data themselves. MLspike is computationally efficient thanks to its original discretization of probability representations; moreover, it can also return spike probabilities or samples. Benchmarked on extensive simulations and real data from seven different preparations, it outperformed state-of-the-art algorithms. Combined with the finding obtained from systematic data investigation (noise level, spiking rate and so on) that photonic noise is not necessarily the main limiting factor, our method allows spike extraction from large-scale recordings, as demonstrated on acousto-optical three-dimensional recordings of over 1,000 neurons in vivo.

The pathogenesis of Parkinson's disease (PD) involves the accumulation of aggregated α-synuclein, which has been suggested to begin in the gastrointestinal tract. Here, we determined the capacity of the appendix to modify PD risk and influence pathogenesis. In two independent epidemiological datasets, involving more than 1.6 million individuals and over 91 million person-years, we observed that removal of the appendix decades before PD onset was associated with a lower risk for PD, particularly for individuals living in rural areas, and delayed the age of PD onset. We also found that the healthy human appendix contained intraneuronal α-synuclein aggregates and an abundance of PD pathology-associated α-synuclein truncation products that are known to accumulate in Lewy bodies, the pathological hallmark of PD. Lysates of human appendix tissue induced the rapid cleavage and oligomerization of full-length recombinant α-synuclein. Together, we propose that the normal human appendix contains pathogenic forms of α-synuclein that affect the risk of developing PD.

The origin of α-synuclein (α-syn)-positive glial cytoplasmic inclusions found in oligodendrocytes in multiple system atrophy (MSA) is enigmatic, given the fact that oligodendrocytes do not express α-syn mRNA. Recently, neuron-to-neuron transfer of α-syn was suggested to contribute to the pathogenesis of Parkinson's disease. In this study, we explored whether a similar transfer of α-syn might occur from neurons to oligodendrocytes, which conceivably could explain how glial cytoplasmic inclusions are formed. We studied oligodendrocytes in vitro and in vivo and examined their ability to take up different α-syn assemblies. First, we treated oligodendrocytes with monomeric, oligomeric, and fibrillar forms of α-syn proteins and investigated whether α-syn uptake is dynamin-dependent. Second, we injected the same α-syn species into the mouse cortex to assess their uptake in vivo. Finally, we monitored the presence of human α-syn within rat oligodendroglial cells grafted in the striatum of hosts displaying Adeno-Associated Virus-mediated overexpression of human α-syn in the nigro-striatal pathway. Here, we show that oligodendrocytes take up recombinant α-syn monomers, oligomers and, to a lesser extent, fibrils in vitro in a concentration and time-dependent manner, and that this process is inhibited by dynasore. Further, we demonstrate in our injection model that oligodendrocytes also internalize α-syn in vivo. Finally, we provide the first direct evidence that α-syn can transfer to grafted oligodendroglial cells from host rat brain neurons overexpressing human α-syn. Our findings support the hypothesis of a neuron-to-oligodendrocyte transfer of α-syn, a mechanism that may play a crucial role in the progression and pathogenesis of MSA.

In Parkinson's disease, intracellular α-synuclein inclusions form in neurons. We suggest that prion-like behavior of α-synuclein is a key component in Parkinson's disease pathogenesis. Although multiple molecular changes are involved in the triggering of the disease process, we propose that neuron-to-neuron transfer is a crucial event that is essential for Lewy pathology to spread from one brain region to another. In this review, we describe key findings in human postmortem brains, cultured cells, and animal models of disease that support the idea that α-synuclein can act as a prion. We consider potential triggers of the α-synuclein misfolding and why the aggregates escape cellular degradation under disease conditions. We also discuss whether different strains of α-synuclein fibrils can underlie differences in cellular and regional distribution of aggregates in different synucleinopathies. Our conclusion is that α-synuclein probably acts as a prion in human diseases, and a deeper understanding of this step in the pathogenesis of Parkinson's disease can facilitate the development of disease-modifying therapies in the future. Dual Perspectives Companion Paper: Parkinson's Disease Is Not Simply a Prion Disorder, by D. James Surmeier, José A. Obeso, and Glenda M. Halliday

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.

Maintenance of genome integrity is a key issue for all living organisms. Cells are constantly exposed to DNA damage due to replication or transcription, cellular metabolic activities leading to the production of Reactive Oxygen Species (ROS) or even exposure to DNA damaging agents such as UV light. However, genomes remain extremely stable, thanks to the permanent repair of DNA lesions. One key mechanism contributing to genome stability is the DNA Damage Response (DDR) that activates DNA repair pathways, and in the case of proliferating cells, stops cell division until DNA repair is complete. The signaling mechanisms of the DDR are quite well conserved between organisms including in plants where they have been investigated into detail over the past 20 years. In this review we summarize the acquired knowledge and recent advances regarding the DDR control of cell cycle progression. Studying the plant DDR is particularly interesting because of their mode of development and lifestyle. Indeed, plants develop largely post-embryonically, and form new organs through the activity of meristems in which cells retain the ability to proliferate. In addition, they are sessile organisms that are permanently exposed to adverse conditions that could potentially induce DNA damage in all cell types including meristems. In the second part of the review we discuss the recent findings connecting the plant DDR to responses to biotic and abiotic stresses.

Abstract Individual identification is a crucial step to answer many questions in evolutionary biology and is mostly performed by marking animals with tags. Such methods are well‐established, but often make data collection and analyses time‐consuming, or limit the contexts in which data can be collected. Recent computational advances, specifically deep learning, can help overcome the limitations of collecting large‐scale data across contexts. However, one of the bottlenecks preventing the application of deep learning for individual identification is the need to collect and identify hundreds to thousands of individually labelled pictures to train convolutional neural networks (CNNs). Here we describe procedures for automating the collection of training data, generating training datasets, and training CNNs to allow identification of individual birds. We apply our procedures to three small bird species, the sociable weaver Philetairus socius, the great tit Parus major and the zebra finch Taeniopygia guttata , representing both wild and captive contexts. We first show how the collection of individually labelled images can be automated, allowing the construction of training datasets consisting of hundreds of images per individual. Second, we describe how to train a CNN to uniquely re‐identify each individual in new images. Third, we illustrate the general applicability of CNNs for studies in animal biology by showing that trained CNNs can re‐identify individual birds in images collected in contexts that differ from the ones originally used to train the CNNs. Finally, we present a potential solution to solve the issues of new incoming individuals. Overall, our work demonstrates the feasibility of applying state‐of‐the‐art deep learning tools for individual identification of birds, both in the laboratory and in the wild. These techniques are made possible by our approaches that allow efficient collection of training data. The ability to conduct individual recognition of birds without requiring external markers that can be visually identified by human observers represents a major advance over current methods.

In all vertebrate species studied thus far, the adult central nervous system harbors neural stem cells that sustain constitutive neurogenesis, as well as latent neural progenitors that can be awakened in lesional contexts. In spite of this common theme, many species differ dramatically in their ability to recruit constitutive progenitors, to awaken latent progenitors, or to enhance or bias neural progenitor fate to achieve successful neuronal repair. This Review summarizes the striking similarities in the essential molecular and cellular properties of adult neural stem cells between different vertebrate species, both under physiological and reparative conditions. It also emphasizes the differences in the reparative process across evolution and how the study of non-mammalian models can provide insights into both basic neural stem cell properties and stimulatory cues shared between vertebrates, and subsequent neurogenic events, which are abortive under reparative conditions in mammals.

BACKGROUND: Cavefish populations belonging to the Mexican tetra species Astyanax mexicanus are outstanding models to study the tempo and mode of adaptation to a radical environmental change. They are currently assigned to two main groups, the so-called "old" and "new" lineages, which would have populated several caves independently and at different times. However, we do not have yet accurate estimations of the time frames of evolution of these populations. RESULTS: We reanalyzed the geographic distribution of mitochondrial and nuclear DNA polymorphisms and we found that these data do not support the existence of two cavefish lineages. Using IMa2, a program that allows dating population divergence in addition to demographic parameters, we found that microsatellite polymorphism strongly supports a very recent origin of cave populations (< 20,000 years). We identified a large number of single-nucleotide polymorphisms (SNPs) in transcript sequences of pools of embryos (Pool-seq) belonging to Pachón cave population and a surface population from Texas. Based on summary statistics that can be computed with this SNP data set together with simulations of evolution of SNP polymorphisms in two recently isolated populations, we looked for sets of demographic parameters that allow the computation of summary statistics with simulated populations that are similar to the ones with the sampled populations. In most simulations for which we could find a good fit between the summary statistics of observed and simulated data, the best fit occurred when the divergence between simulated populations was less than 30,000 years. CONCLUSIONS: Although it is often assumed that some cave populations have a very ancient origin, a recent origin of these populations is strongly supported by our analyses of independent sets of nuclear DNA polymorphism. Moreover, the observation of two divergent haplogroups of mitochondrial and nuclear genes with different geographic distributions support a recent admixture of two divergent surface populations, before the isolation of cave populations. If cave populations are indeed only several thousand years old, many phenotypic changes observed in cavefish would thus have mainly involved the fixation of genetic variants present in surface fish populations and within a very short period of time.

Objective Chronic alcohol consumption is an important cause of liver-related deaths. Specific intestinal microbiota profiles are associated with susceptibility or resistance to alcoholic liver disease in both mice and humans. We aimed to identify the mechanisms by which targeting intestinal microbiota can improve alcohol-induced liver lesions. Design We used human associated mice, a mouse model of alcoholic liver disease transplanted with the intestinal microbiota of alcoholic patients and used the prebiotic, pectin, to modulate the intestinal microbiota. Based on metabolomic analyses, we focused on microbiota tryptophan metabolites, which are ligands of the aryl hydrocarbon receptor (AhR). Involvement of the AhR pathway was assessed using both a pharmacological approach and AhR-deficient mice. Results Pectin treatment modified the microbiome and metabolome in human microbiota-associated alcohol-fed mice, leading to a specific faecal signature. High production of bacterial tryptophan metabolites was associated with an improvement of liver injury. The AhR agonist Ficz (6-formylindolo (3,2-b) carbazole) reduced liver lesions, similarly to prebiotic treatment. Conversely, inactivation of the ahr gene in alcohol-fed AhR knock-out mice abrogated the beneficial effects of the prebiotic. Importantly, patients with severe alcoholic hepatitis have low levels of bacterial tryptophan derivatives that are AhR agonists. Conclusions Improvement of alcoholic liver disease by targeting the intestinal microbiota involves the AhR pathway, which should be considered as a new therapeutic target.

The mammalian nervous system is comprised of a seemingly infinitely complex network of specialized synaptic connections that coordinate the flow of information through it. The field of connectomics seeks to map the structure that underlies brain function at resolutions that range from the ultrastructural, which examines the organization of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, distant and local connections between neuronal populations are identified, providing insights into circuit-level architecture. Although neural tract tracing techniques have been available to experimental neuroscientists for many decades, considerable methodological advances have been made in the last 20 years due to synergies between the fields of molecular biology, virology, microscopy, computer science and genetics. As a consequence, investigators now enjoy an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to identify their efferent and afferent connectomes. Unfortunately, the intersectional nature of this progress presents newcomers to the field with a daunting array of technologies that have emerged from disciplines they may not be familiar with. This review outlines the current state of mesoscale connectomic approaches, from data collection to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used by contemporary neuroscientists to resolve mesoscale organization, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. We consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing data and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.

Microglia have been discovered to undergo repopulation following ablation. However, the functionality of repopulated microglia and the mechanisms regulating microglia repopulation are unknown. We examined microglial homeostasis in the adult mouse retina, a specialized neural compartment containing regular arrays of microglia in discrete synaptic laminae that can be directly visualized. Using in vivo imaging and cell-fate mapping techniques, we discovered that repopulation originated from residual microglia proliferating in the central inner retina that subsequently spread by centrifugal migration to fully recapitulate pre-existing microglial distributions and morphologies. Repopulating cells fully restored microglial functions including constitutive "surveying" process movements, behavioral and physiological responses to retinal injury, and maintenance of synaptic structure and function. Microglial repopulation was regulated by CX3CL1-CX3CR1 signaling, slowing in CX3CR1 deficiency and accelerating with exogenous CX3CL1 administration. Microglial homeostasis following perturbation can fully recover microglial organization and function under the regulation of chemokine signaling between neurons and microglia.

The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.

Accruing evidence suggests that prion-like behavior of fibrillar forms of α-synuclein, β-amyloid peptide and mutant huntingtin are responsible for the spread of the lesions that characterize Parkinson disease, Alzheimer disease and Huntington disease, respectively. It is unknown whether these distinct protein assemblies are transported within and between neurons by similar or distinct mechanisms. It is also unclear if neuronal death or injury is required for neuron-to-neuron transfer. To address these questions, we used mouse primary cortical neurons grown in microfluidic devices to measure the amounts of α-synuclein, Aβ42 and HTTExon1 fibrils transported by axons in both directions (anterograde and retrograde), as well as to examine the mechanism of their release from axons after anterograde transport. We observed that the three fibrils were transported in both anterograde and retrograde directions but with strikingly different efficiencies. The amount of Aβ42 fibrils transported was ten times higher than that of the other two fibrils. HTTExon1 was efficiently transported in the retrograde direction but only marginally in the anterograde direction. Finally, using neurons from two distinct mutant mouse strains whose axons are highly resistant to neurodegeneration (Wld(S) and Sarm1(-/-)), we found that the three different fibrils were secreted by axons after anterograde transport, in the absence of axonal lysis, indicating that trans-neuronal spread can occur in intact healthy neurons. In summary, fibrils of α-synuclein, Aβ42 and HTTExon1 are all transported in axons but in directions and amounts that are specific of each fibril. After anterograde transport, the three fibrils were secreted in the medium in the absence of axon lysis. Continuous secretion could play an important role in the spread of pathology between neurons but may be amenable to pharmacological intervention.

Abstract This review describes the duality between color and kinematics and its applications, with the aim of gaining a deeper understanding of the perturbative structure of gauge and gravity theories. We emphasize, in particular, applications to loop-level calculations, the broad web of theories linked by the duality and the associated double-copy structure, and the issue of extending the duality and double copy beyond scattering amplitudes. The review is aimed at doctoral students and junior researchers both inside and outside the field of amplitudes and is accompanied by various exercises.