Max Planck Institute of Neurobiology

Abstract The transition from well-fed to food-deprived conditions in C. elegans triggers a stereotypic exploration behaviour in C. elegans characterised by a temporal decrease in cumulative reorientations. We conducted a screen of neuropeptide mutants and identified several candidates involved in modulating this behaviour. Among these, the neuropeptide FLP-15 emerged as a key regulator. Our observations revealed that FLP-15 regulates the frequency of reversals during foraging through the I2 pharyngeal neuron via the G-protein coupled receptor NPR-3. Mutants lacking either flp-15, npr-3 or both displayed a significant defect in reversal frequency which did not decline temporally unlike in wild-type animals. This study also describes the expression pattern of NPR-3 in a subset of head neurons, predominantly comprising of dopaminergic neurons. Finally, flp-15 expression studies and exogenous dopamine supplementation assays revealed that FLP-15 may regulate exploratory search by modulating dopamine transmission, highlighting a novel neuropeptide-dopamine interaction involved in the control of foraging behaviours.

Early-onset SCN2A developmental and epileptic encephalopathy is caused by SCN2A gain-of-function variants. Here we describe the clinical experience with intrathecally administered elsunersen, a gapmer antisense oligonucleotide targeting SCN2A, in a female preterm infant with early-onset SCN2A developmental and epileptic encephalopathy, in an expanded access program. Before elsunersen treatement, the patient was in status epilepticus for 7 weeks with a seizure frequency of 20-25 per hour. Voltage-clamp experiments confirmed impaired channel inactivation and increased persistent current consistent with a gain-of-function mechanism. Elsunersen treatment demonstrated a favorable safety profile with no severe or serious adverse events reported after 19 intrathecal administrations over 20 months. After administration in combination with sodium channel blockers, status epilepticus was interrupted intermittently and ultimately ceased after continued dosing. A >60% reduction in seizure frequency corresponding to five to seven seizures per hour was observed, which has been sustained during follow-up until the age of 22 months. These data provide preliminary insights on the safety and efficacy of elsunersen in a preterm infant. Additional investigation on the benefits of elsunersen in clinical trials is warranted.

Abstract Heterobifunctional small molecules recruiting the proteasomal machinery enable the targeted protein degradation (TPD) of selected proteins and considerably broaden the druggable space for therapeutic use. Many such molecules have seen fantastic success in preclinical and clinical developments, e.g., for targeted degradation of specific oncoproteins that were previously inaccessible for cancer therapy. Even though early hits are quickly generated by adapting the modular concept of bifunctional degraders, most of such molecules are against the central rules of a “drug-like” molecule: they are usually larger in size (800-1500 Da) and often lack sufficient solubility. These early bifunctional degraders hits require exhaustive structure optimization in each case to optimize pharmacokinetics (PK) profiles and bioavailability. Here, we report Tubulis’ antibody-degrader conjugates that exploit bifunctional degraders as high-potency bioactive payloads to be released specifically inside cells that are targeted by the antibody. Our linker technology ensures high degrader-to-antibody ratios (DARs) and excellent linker stability in blood plasma. Moreover, we liberate the degrader after receptor-mediated uptake of the ADC and show high efficiencies in target protein degradation. Most importantly, our conjugates exhibit antibody-like PK properties that ensure long-lasting plasma circulation of high DAR constructs over weeks, even when bifunctional degraders are employed that have not been optimized for PK properties. Taken together, these factors allow impressive in vivo efficacy of the conjugates after single dose application in preclinical animal models. Citation Format: Jan G. Felber, Anil P. Jagtap, Philipp Ochtrop, Saskia Schmitt, Isabelle Mai, Sarah Herterich, Paul Machui, Dietmar König, Dominik simon vogt, Schumacher, Jonas Helma-Smets, Annette Vogl, Marc-André Kasper. Antibody-degrader conjugates unlock the therapeutic potential of heterobifunctional degraders through antibody-like pharmacokinetics and excellent in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7018.

During prey pursuit, how eye rotations, such as saccades, enable continuous tracking of erratically moving targets while enabling an animal to navigate through the environment is unknown. To better understand this, we measured head and eye rotations in freely running ferrets during pursuit behavior. By also tracking the target and all environmental features, we reconstructed the animal's visual fields and their relationship to retinal structures. In the reconstructed visual fields, the target position clustered on and around the high-acuity retinal area location, the area centralis, and surprisingly, this cluster was not significantly shifted by digital removal of either eye saccades, exclusively elicited when the ferrets made turns, or head rotations that were tightly synchronized with the saccades. Here, we show that, while the saccades did not fixate the moving target with the area centralis, they instead aligned the area centralis with the intended direction of travel. This also aligned the area centralis with features of the optic flow pattern, such as flow direction and focus of expansion, used for navigation by many species. While saccades initially rotated the eyes in the same direction as the head turn, saccades were followed by eye rotations countering the ongoing head rotation, which reduced image blur and limited information loss across the visual field during head turns. As we measured the same head and eye rotational relationship in freely moving tree shrews, rats, and mice, we suggest that these saccades and counter-rotations are a generalized mechanism enabling mammals to navigate complex environments during pursuit.

The translation of mRNA into proteins in multicellular organisms needs to be carefully tuned to changing proteome demands in development and differentiation, while defects in translation often have a disproportionate impact in distinct cell types. Here we used inducible CRISPR interference screens to compare the essentiality of genes with functions in mRNA translation in human induced pluripotent stem cells (hiPS cells) and hiPS cell-derived neural and cardiac cells. We find that core components of the mRNA translation machinery are broadly essential but the consequences of perturbing translation-coupled quality control factors are cell type dependent. Human stem cells critically depend on pathways that detect and rescue slow or stalled ribosomes and on the E3 ligase ZNF598 to resolve a distinct type of ribosome collision at translation start sites on endogenous mRNAs with highly efficient initiation. Our findings underscore the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.

Abstract Thylakoid membranes coordinate the light reactions of photosynthesis across multiple scales, coupling the architecture of an elaborate membrane network to the spatial organization of individual protein complexes embedded within this network. Previously, we used in situ cryo-electron tomography (cryo-ET) to reveal the native thylakoid architecture of the green alga Chlamydomonas reinhardtii [1] and then map the molecular organization of these thylakoids with single-molecule precision [2]. However, it remains to be shown how generalizable this green algal blueprint is to the thylakoids of vascular plants, which possess distinct membrane architecture subdivided into grana stacks interconnected by non-stacked stromal lamellae. Here, we continue our cryo-ET investigation to reveal the molecular architecture of thylakoids within intact chloroplasts isolated from spinach ( Spinacia oleracea ). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply and further develop AI-based computational approaches for automated membrane segmentation and membrane protein picking [3], enabling us to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that, despite different 3D architecture, the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. In contrast to isolated plant thylakoids, where semi-crystalline arrays of photosystem II (PSII) appear to hold some membranes together, we find in intact chloroplasts that PSII is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to C. reinhardtii , we observe strict lateral heterogeneity of PSII and PSI at the boundary between appressed and non-appressed thylakoid domains, with no evidence for a distinct grana margin region where these complexes have been proposed to intermix. Based on these measurements, we support a simple two-domain model for the molecular organization of thylakoid membranes in both green algae and plants.

Abstract Active and passive sensing strategies are integral to an animal’s behavioral repertoire. Nevertheless, there is a lack of information regarding the neuronal circuitry that underpins these strategies, particularly at the thalamus level. We evaluated how active versus passive whisker deflections are represented in single neurons of the ventral posterior thalamus (VPM) and the posterior medial thalamus (POm) in awake mice. These are the first-and higher-order thalamic nuclei of the whisker system, respectively. VPM neurons robustly responded to both active and passive whisker deflections, while POm neurons showed a preference for passive deflections and responded poorly to active touches. This response disparity could not be explained by the animal’s voluntary whisking state or stimulus kinetics. In contrast, cortical activity significantly influenced POm’s responses to passive touch. Inhibition of the barrel cortex strongly attenuated whisker responses in POm and simultaneously increased the whisking phase coding. This suggests that POm receives touch information from the cortex and phase information from the brainstem. Together, these findings suggest two thalamic relay streams, where VPM robustly relays both active and passive deflection, while POm’s sensitivity requires top-down cortical involvement to signal salient events such as unexpected passive deflections.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Abstract While immune checkpoint therapies have revolutionized cancer treatment, their efficacy remains limited in colorectal cancer (CRC) and other malignancies. Tumors employ diverse strategies to evade immune-mediated destruction, hindering the success of immunotherapies. We introduce iOTarg, a robust high-throughput screening platform designed to uncover resistance pathways against T cell-mediated tumor killing within the tumor microenvironment. Focusing on CRC, we utilized iOTarg to dissect evasion mechanisms against antigen-specific T cell (TC) killing. In a representative CRC cell line co-culture system (SW480, HCT116 or COLO-678) with various TC sources, we gained key insights of tumor resistance towards killing. Survivin-specific TCs induce strong tumor lysis while tumor-infiltrating lymphocyte 412 (TIL412) represent a weaker basal killing representative of the situation in the tumor. Knockout of β₂-microglobulin abrogated TC-mediated killing, while c-FLIP knockout significantly enhanced tumor lysis. Intriguingly, PD-L1 blockade had no impact on TC activation or killing, underlining its lack of efficacy in CRC. Screening over 6000 druggable genes revealed several critical candidates, including anti-apoptotic BIRC family members (BIRC2, 3), apoptosis regulators (Bcl-xL, XIAP), TNF signaling regulators (TRAF2, MAP3K7), and genes recently described as sensitization mediators (B3GNT3, CHMP4B). Caspase-8 knockout prevented TC-mediated killing of CRC cells, confirming its crucial role in immune-mediated tumor cell clearance. Further validation in secondary screenings reinforced a subset of genes as strong regulators of tumor cell killing, uncovering an array of CRC resistance mechanisms. Our findings present a pioneering platform delineating resistance mechanisms against T cell-mediated colorectal tumor killing. We identify established and novel targets, offering prospects for novel therapeutics and expanding immune oncology treatment options. This platform holds promise for developing first-in-class therapies targeting the tumor microenvironment, potentially transforming CRC treatment paradigms. Citation Format: Tillmann Michels, Valentina Volpin, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Aleksandra Weglarz, Kilian Baritz, Kritika Sudan, Szilvia Bak-Kiss, Alina Huth, Carolin Strobl, Adriana Turqueti-Neves, Nisit Khandelwal. iOTarg screening platform reveals novel mechanisms of colorectal cancer evasion from antigen-specific tumor cell killing by T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB080.