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Schlegel, P.

Publications and source records attributed to Schlegel, P..

At least 19 recordsLinked to original sources

Latent generative search unlocks de novo design of untapped biomolecular interactions at scale

De novo protein design has advanced rapidly, yet designing binders to polar, solvent-exposed epitopes and small, flexible ligands remains challenging. Such hydrated surfaces and flexible molecules, including carbohydrates, provide few of the hydrophobic contacts favoured by current methods and have largely resisted de novo binders. To address this challenge, here we introduce latent generative search for binder design, a novel framework that uses reward-guided search at inference time to steer the Proteina-Complexa generative model. The model codesigns sequence and structure - generating them together in a continuous latent space - and thereby removes the inverse-folding step on which current methods rely. In a screen of more than one million designs by multiplexed phage display, latent generative search produced more validated binders than every other method tested, its codesigned sequences surpassing post hoc redesign. It delivered high-affinity binders across therapeutic receptors, a viral attachment protein and intracellular signalling targets. Our approach also accessed previously untapped biology, generating the first de novo proteins that bind a free carbohydrate, including one that discriminates between blood-group antigens - a polar, flexible target class beyond the reach of current design methods.

bioengineering↗

Structured Connectivity Across Multiple Drosophila Mushroom Bodies

Learning and memory depend on plasticity within neural circuits, but the extent to which the underlying architecture of memory networks is stereotyped or structured remains unclear. Random connectivity may maximise stimulus discrimination, whereas structured connectivity could preferentially route behaviourally relevant information. Here, we compare five adult Drosophila melanogaster Mushroom Body (MB) hemispheres across three connectomes and establish a unified cross-connectome framework for uniglomerular projection neurons (uPNs), Kenyon Cells (KCs), Mushroom Body Output Neurons (MBONs) and Lateral Horn Centrifugal Neurons (LHCENTs). Despite variation in KC abundance and fine-scale connectivity, we identify reproducible organisation across the uPN[->]KC and KC[->]MBON pathways. Food-associated uPN channels consistently contact a greater proportion of KCs than expected by chance, and this bias is preferentially routed through KCs towards approach-promoting MBONs. Sensory input also remains stereotyped across sister MBONs from the same hemisphere despite variation in their individual KC partners. Finally, recurrent LHCENT feedback (predicted-inhibitory) preferentially targets KCs receiving these food-associated inputs. Together, these findings show that a memory network can accommodate local variability while preserving structured pathways that prioritise ethologically relevant sensory information.

neuroscience↗

A generalizable interface-seeded framework for de novo design of functional oligomers

Protein oligomers are ubiquitous in biological systems and essential for function. However, the de novo design of oligomers that controllably assemble in response to exogenous stimuli remains challenging. Here, we present an AI-based generative approach that leverages an interface-seeded strategy for designing responsive homo-oligomers from isolated interaction modules. Experimentally validated designs are highly accurate and explore new-to-nature topologies. We show that designs effectively respond to their chemical triggers with conditional oligomerization or to phosphorylation-driven conformational changes with reversible oligomerization. We further functionalized our responsive assemblies to build ligand-dependent membrane binding systems and phosphorylation-controlled gene regulatory switches. Our framework enables the generalizable design of responsive protein complexes, opening novel possibilities for the engineering of biosynthetic systems with sophisticated regulatory mechanisms.

biochemistry↗

An aorta to insulin producing cell sensor for internal state in Drosophila

The cellular circuits through which the internal state of an organism is sensed and relayed to the brain, and through which they bring about appropriate physiological and behavioral responses, remain largely unelucidated. Using whole-animal electron microscopy reconstruction of a Drosophila larva, we previously identified an Aorta sensory organ whose somata lie on the periphery of the brain, with dendritic arborizations in the wall of the anterior aorta, and whose axons project to the protocerebrum and synapse onto the insulin-producing cells (IPCs). Here we characterize the full anatomy and connectivity of this sensory organ. We show that the aorta sensory neurons make their strongest direct synaptic contacts onto IPCs and the neurosecretory cells producing Dromyosuppressin (DMS), with only weaker direct contacts to diuretic hormone 44 (DH44). They also make direct contacts to the enteric serotonergic neurons modulating swallowing (Se0ens), and to two single mushroom body neurons, DAN-j1 and MBON-d3, of the learning and memory center of Drosophila. Calcium imaging shows that the neurons are activated by fructose, and optogenetic manipulation reveals that their activity influences peptide content in IPCs, DH44 and DMS cells. Polysynaptic paths to the brain run through two morphologically distinct interneuron families. One, formed by the Hugin protocerebral neurons, targets the neuroendocrine system and is associated with bitter taste and pathogen induced feeding suppression. The other, formed by the BAmas projection neurons, targets dopaminergic input neurons and output neurons of the mushroom body. The Aorta sensory pathway therefore couples internal hemolymph state to two parallel central systems -- endocrine state control and mushroom body-associated value updating -- and provides a candidate substrate for a metabolic memory through which post-ingestive state is fed back into future feeding choice.

neuroscience↗

Computational design and experimental characterization of mini-protein binders targeting Nipah, Langya and Measles virus receptor-binding domains

A lack of reagents represents a major bottleneck in pandemic preparedness and rapid vaccine development. It is therefore important to enable the design of reagents for use in the treatment and diagnosis of emerging viral diseases. Ideally, the design and identification platform is fast, can be performed by testing only a small number of candidates and enables a generally applicable strategy. In this study, we assessed the ability of recently developed computational protein design tools to establish such a workflow for validating paramyxovirus receptor-binding protein de novo binders as such reagents. The family Paramyxoviridae includes various members that cause severe disease and exhibit re-occurring zoonotic spillover events, with documented human infections over the past decades. We successfully designed, identified, and characterized mini-proteins targeting the receptor binding proteins of Nipah virus, Langya virus, and Measles virus while screening as few as 10-16 designs per target. The resulting functional binders have moderate to low nanomolar affinities and display high on-target specificity. We further showed that our most promising Nipah virus receptor-binding protein binder is able to inhibit human receptor binding in vitro and competes for an epitope that overlaps with that of the neutralizing antibody HENV-117. However, despite these promising results, this Nipah binder is only weakly neutralizing, preventing therapeutic applications. Nevertheless, we established a platform, applicable to rapidly generate diagnostically relevant proteins from only a small number of candidates, and developed novel reagents for the Paramyxoviridae family.

bioinformatics↗

Loss of noradrenergic Fkbp5 disrupts social behavior and norepinephrine dynamics in the basolateral amygdala

Social dysfunction is common in depression and varies with stress exposure and genetic risk. The current study identifies a cell-type specific role for Fkbp5, a glucocorticoid receptor co-chaperone, in noradrenergic neurons engaged during social stress. Acute social stress upregulated Fkbp5 in the locus coeruleus (LC), whereas repeated exposure attenuated this effect. Noradrenergic Fkbp5 deletion (Fkbp5Nat) increased pro-social behavior exclusively in male mice. In the basolateral amygdala (BLA), social interaction reduced norepinephrine (NE) turnover in wild-type but not Fkbp5Nat mice. Consistently, proteomics revealed mitochondrial/energy and synapse-related remodeling in BLA neurons. Miniscope imaging showed that behavior-locked NE transients in BLA were selectively blunted in Fkbp5Nat mice during interaction with outbred CD1 conspecifics, while same-strain C57BL/6N encounters preserved NE dynamics. Together, this study indicates that Fkbp5 tunes LC-BLA output to social salience in a sex- and context-dependent manner, suggesting a circuit-specific route to normalize social salience without broadly suppressing noradrenergic function.

neuroscience↗

Sexual dimorphism in the complete connectome of the Drosophila male central nervous system

Sex differences in behaviour exist across all animals, typically under strong genetic regulation. In Drosophila, fruitless/doublesex transcription factors can identify dimorphic neurons but their organisation into functional circuits remains unclear. We present the connectome of the entire Drosophila male central nervous system. This contains 166,691 neurons spanning the brain and nerve cord, fully proofread and annotated including fruitless/doublesex expression and 11,691 types. We provide the first comprehensive comparison between male and female brain connectomes to synaptic resolution, finding 7,205 isomorphic, 114 dimorphic, 262 male-specific and 69 female-specific types. This resource enables analysis of full sensory-to-motor circuits underlying complex behaviours and the impact of dimorphic elements. Sex-specific/dimorphic neurons are concentrated in higher brain centres while the sensory and motor periphery are largely isomorphic. Within higher centres, male-specific connections are organised into hotspots defined by male-specific neurons or arbours. Numerous circuit switches reroute sensory information to form antagonistic circuits controlling opposing behaviours.

neuroscience↗

From Sensory Detection to Motor Action: The Comprehensive Drosophila Taste-Feeding Connectome

Gustatory systems drive critical survival behaviors such as feeding, foraging, and social interactions. However, gustation remains one of the least mapped sensory modalities at the connectome level. Here, we present the first complete wiring diagram of the male Drosophila adult gustatory system, comprehensively reconstructing gustatory receptor neurons (GRNs) from peripheral organs in a contiguous electron microscopy volume spanning brain, cervical connective, and ventral nerve cord. Integrating this with existing datasets, we generated a pan-CNS, cross-sex connectome that reveals GRN diversity through connectivity-based clustering, molecular identity mapping, and sexual dimorphism analysis. We mapped all feeding motor neurons and traced complete sensory-to-motor pathways to feeding, foraging, endocrine, and social behavior circuits. The emerging circuit architectures reveal distinct circuits for nutrient assessment, motor control, neuroendocrine regulation, and courtship. This work defines the gustatory systems organization at synaptic resolution and provides a framework for understanding how internal states modulate sensory-driven decisions across behavioral contexts.

neuroscience↗

Sexually-dimorphic neurons in the Drosophila whole-brain connectome

Sexual dimorphisms in the brain arise from differences in cell number, morphology, and connectivity, and underlie sex-specific behaviors. In Drosophila, these differences are largely governed by the transcription factors Fruitless (Fru) and Doublesex (Dsx), expressed in neurons implicated in reproductive and social behaviors. However, how these neurons are integrated into the connectome to drive different behaviors in different sexes remains unclear. Here, we identify 98 putative Fru- or Dsx-expressing cell types ([~]1700 neurons) in the female whole-brain connectome by matching electron microscopy reconstructions to light microscopy images. We then analyze their connectivity within the whole-brain network. Fru/Dsx neurons are strongly interconnected, but are not part of a private network. We measure distances to the sensory periphery to examine multisensory interactions, and map connections to descending neurons that drive behavior. We further cluster Fru/Dsx neurons based on whole-brain connectivity, and uncover functional groupings. We analyze a subset of strongly connected Fru/Dsx neurons to make functional predictions, and test some of these with experiments. Together, this work provides a brain-wide framework for understanding the organization of sexually dimorphic circuits and establishes a resource for dissecting the neural basis of social and sexual behaviors.

neuroscience↗

HyperMPNN - A general strategy to design thermostable proteins learned from hyperthermophiles

Stability is a key factor to enable the use of recombinant proteins in therapeutic or biotechnological applications. Deep learning protein design approaches like ProteinMPNN have shown strong performance both in creating novel proteins or stabilizing existing ones. However, it is unlikely that the stability of the designs will significantly exceed that of the natural proteins in the training set, which are biophysically only marginally stable. Therefore, we collected predicted protein structures from hyperthermophiles, which differ substantially in their amino acid composition from mesophiles. Notably, ProteinMPNN fails to recover their unique amino acid composition. Here we show that a retrained network on predicted proteins from hyperthermophiles, termed HyperMPNN, not only recovers this unique amino acid composition but can also be applied to proteins from non-hyperthermophiles. Using this novel approach on a protein nanoparticle with a melting temperature of 65{degrees}C resulted in designs remaining stable at 95{degrees}C. In conclusion, we created a new way to design highly thermostable proteins through self-supervised learning on data from hyperthermophiles.

bioinformatics↗

Comparative connectomics of the descending and ascending neurons of the Drosophila nervous system: stereotypy and sexual dimorphism

In most complex nervous systems there is a clear anatomical separation between the nerve cord, which contains most of the final motor outputs necessary for behaviour, and the brain. In insects, the neck connective is both a physical and information bottleneck connecting the brain and the ventral nerve cord (VNC, spinal cord analogue) and comprises diverse populations of descending (DN), ascending (AN) and sensory ascending neurons, which are crucial for sensorimotor signalling and control. Integrating three separate EM datasets, we now provide a complete connectomic description of the ascending and descending neurons of the female nervous system of Drosophila and compare them with neurons of the male nerve cord. Proofread neuronal reconstructions have been matched across hemispheres, datasets and sexes. Crucially, we have also matched 51% of DN cell types to light level data defining specific driver lines as well as classifying all ascending populations. We use these results to reveal the general architecture, tracts, neuropil innervation and connectivity of neck connective neurons. We observe connected chains of descending and ascending neurons spanning the neck, which may subserve motor sequences. We provide a complete description of sexually dimorphic DN and AN populations, with detailed analysis of circuits implicated in sex-related behaviours, including female ovipositor extrusion (DNp13), male courtship (DNa12/aSP22) and song production (AN hemilineage 08B). Our work represents the first EM-level circuit analyses spanning the entire central nervous system of an adult animal.

neuroscience↗

Network Statistics of the Whole-Brain Connectome of Drosophila

Brains comprise complex networks of neurons and connections. Network analysis applied to the wiring diagrams of brains can offer insights into how brains support computations and regulate information flow. The completion of the first whole-brain connectome of an adult Drosophila, the largest connectome to date, containing 130,000 neurons and millions of connections, offers an unprecedented opportunity to analyze its network properties and topological features. To gain insights into local connectivity, we computed the prevalence of two- and three-node network motifs, examined their strengths and neurotransmitter compositions, and compared these topological metrics with wiring diagrams of other animals. We discovered that the network of the fly brain displays rich club organization, with a large population (30% percent of the connectome) of highly connected neurons. We identified subsets of rich club neurons that may serve as integrators or broadcasters of signals. Finally, we examined subnetworks based on 78 anatomically defined brain regions or neuropils. These data products are shared within the FlyWire Codex and will serve as a foundation for models and experiments exploring the relationship between neural activity and anatomical structure.

neuroscience↗

Neuronal wiring diagram of an adult brain

Connections between neurons can be mapped by acquiring and analyzing electron microscopic (EM) brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative, yet inadequate for understanding brain function more globally. Here, we present the first neuronal wiring diagram of a whole adult brain, containing 5x107 chemical synapses between [~]130,000 neurons reconstructed from a female Drosophila melanogaster. The resource also incorporates annotations of cell classes and types, nerves, hemilineages, and predictions of neurotransmitter identities. Data products are available by download, programmatic access, and interactive browsing and made interoperable with other fly data resources. We show how to derive a projectome, a map of projections between regions, from the connectome. We demonstrate the tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine, and descending neurons), across both hemispheres, and between the central brain and the optic lobes. Tracing from a subset of photoreceptors all the way to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviors. The technologies and open ecosystem of the FlyWire Consortium set the stage for future large-scale connectome projects in other species.

neuroscience↗

A consensus cell type atlas from multiple connectomes reveals principles of circuit stereotypy and variation

The fruit fly Drosophila melanogaster combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the flys success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper1, this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this [~]130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database2. Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the "hemibrain" connectome3. In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

neuroscience↗

Systematic annotation of a complete adult male Drosophila nerve cord connectome reveals principles of functional organisation

Nervous systems function as ensembles of neurons communicating via synaptic connections, and a functional understanding of nervous systems requires extensive knowledge of their connectomes. In a companion paper (Takemura et al., 2023), we describe the acquisition of a complete fruit fly nerve cord connectome, the first for an animal that can walk or fly. Here, to efficiently navigate and to appreciate the biological significance of this connectome, we categorise and name nearly all neurons systematically and link them to the experimental literature. We employ a system of hierarchical coarse annotations and group similar neurons across the midline and across segments, then define systematic cell types for sensory neurons, intrinsic neurons, ascending neurons, and non-motor efferent neurons. Stereotyped arrays of neuroblasts generate related neuron populations called hemilineages that repeat across the segments of the nerve cord. We confirm that larval-born neurons from a given hemilineage generally express the same neurotransmitter but find that earlier born neurons often express a different one. We match over 35% of intrinsic, ascending, and non-motor efferent neurons across segments, defining serial sets which were crucial for systematic typing of motor neurons and sensory neurons. We assign a sensory modality to over 5000 sensory neurons, cluster them by connectivity, and identify serially homologous cell types and a layered organisation likely corresponding to peripheral topography. Finally, we present selected examples of sensory circuits predicated on programmatic analysis of a complete VNC connectome. Our annotations are critical for analysing the structure of descending input to the nerve cord and of motor output, both described in a third companion paper (Cheong et al., 2023). These annotations are being released as part of the neuprint.janelia.org and clio.janelia.org web applications and also serve as the basis for programmatic analysis of the connectome through dedicated tools that we describe in this paper.

neuroscience↗

A Connectome of the Male Drosophila Ventral Nerve Cord

Animal behavior is principally expressed through neural control of muscles. Therefore understanding how the brain controls behavior requires mapping neuronal circuits all the way to motor neurons. We have previously established technology to collect large-volume electron microscopy data sets of neural tissue and fully reconstruct the morphology of the neurons and their chemical synaptic connections throughout the volume. Using these tools we generated a dense wiring diagram, or connectome, for a large portion of the Drosophila central brain. However, in most animals, including the fly, the majority of motor neurons are located outside the brain in a neural center closer to the body, i.e. the mammalian spinal cord or insect ventral nerve cord (VNC). In this paper, we extend our effort to map full neural circuits for behavior by generating a connectome of the VNC of a male fly.

neuroscience↗

Serotonergic reinforcement of a complete swallowing circuit

How the body interacts with the brain to perform vital life functions such as feeding is one of the fundamental questions in physiology and neuroscience. Here, we use a whole-animal scanning transmission electron microscopy dataset of Drosophila to map out the neuronal circuits that connect the entire enteric nervous system to the brain via the insect vagus nerve at synaptic resolution. This revealed a periphery-brain feedback loop in which Piezo-expressing mechanosensory neurons sense food intake and convey that information onto serotonergic neurons within the brain. These serotonergic neurons integrate the interoceptive information with external and central inputs, and in turn stabilize rhythmic activity of serotonin receptor 7 expressing peripheral motor neurons that drive swallowing. Strikingly, the very same motor neurons also share an efference copy of their activity with the aforementioned mechanosensory neurons, thereby closing the motor-sensory-modulatory loop. Our analysis elucidates an elemental, albeit surprisingly complex reinforcement circuit in which rhythmic motor patterns are stabilized through afferent signaling to central serotonergic neurons upon completion of a rewarding action. The circuit motif is constructed to allow the distinction between self-generated action and those in response to the environment.

neuroscience↗

A leaky integrate-and-fire computational model based on the connectome of the entire adult Drosophila brain reveals insights into sensorimotor processing

The forthcoming assembly of the adult Drosophila melanogaster central brain connectome, containing over 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain. Here, we create a leaky integrate-and-fire computational model of the entire Drosophila brain, based on neural connectivity and neurotransmitter identity, to study circuit properties of feeding and grooming behaviors. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation. Computational activation of neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing, a testable hypothesis that we validate by optogenetic activation and behavioral studies. Moreover, computational activation of different classes of gustatory neurons makes accurate predictions of how multiple taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Our computational model predicts that the sugar and water pathways form a partially shared appetitive feeding initiation pathway, which our calcium imaging and behavioral experiments confirm. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit that do not overlap with gustatory circuits, and accurately describes the circuit response upon activation of different mechanosensory subtypes. Our results demonstrate that modeling brain circuits purely from connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can accurately describe complete sensorimotor transformations.

neuroscience↗