Nanoscale Microscopy and Molecular Physiology of the Brain Cluster of Excellence 171 — DFG Research Center 103

Summary The non-templated addition of the 3’CCA end is the final universal step of tRNA maturation. In humans, 3’CCA addition on nuclear- (nu-tRNA) and mitochondria-encoded tRNAs (mt-tRNA) is catalyzed by a single CCA-adding enzyme, TRNT1, but its precise mechanism remains unknown. Here, we report structures of TRNT1 trapped at various stages during the 3’CCA addition cycle on canonical and non-canonical mt-tRNAs bound to the mitochondrial tRNA maturation platform TRMT10C-SDR5C1. Combined with biochemical data, these structures demonstrate that 3’CCA addition proceeds by a continuous polymerization and translocation mechanism, in which the growing RNA primer remodels the TRNT1 catalytic site to define the specificity of non-templated 3’CCA addition. Moreover, they reveal a relaxed recognition mode that allows TRNT1 to mature both canonical nu-tRNA and non-canonical mt-tRNA substrates. Finally, biochemical analyses of disease-associated TRNT1 variants provide insights into their molecular pathogenesis. Taken together, these results provide a detailed mechanistic picture of human tRNA-3’CCA maturation.

Peroxidasin (PXDN), an extracellular matrix (ECM)-associated peroxidase, has been implicated in cancer progression. However, its roles in melanoma biology and therapeutic sensitivity remain unclear. Here, we demonstrate that elevated PXDN expression is associated with poor prognosis and reduced survival in melanoma patients. Functional studies revealed that PXDN depletion impairs melanoma cell proliferation, disrupts the cell cycle, and reduces melanoma cell invasive capacities. Furthermore, we found that secreted PXDN modulates anti-melanoma immunity by enhancing melanoma resistance to natural killer (NK)-cell-mediated cytotoxicity. Structural modeling identified a trimeric organization of PXDN, stabilized by disulfide-linked peroxidase domains. Molecular dynamics simulations identified a previously unknown inhibitory interaction between the PXDN N-terminal leucine-rich repeat domain and the NK cell-activating receptor NKG2-D type II integral membrane protein (NKG2D). These findings uncover a redox-independent role for PXDN in promoting immune evasion and tumor progression. Overall, our study highlights PXDN as a critical regulator of melanoma cell biology and a potential therapeutic target for NK-cell-based immunotherapy in melanoma and other solid cancers.

ABSTRACT Laser ablation is a widely used technique for the fabrication of periodic structures on solids to manipulate their surface properties. A fundamental challenge in the formation of laser‐induced periodic surface structures (LIPSS) is ascertaining the correlation between laser parameters and LIPSS characteristics. This study focuses on the characterization of LIPSS in terms of the period of low spatial frequency LIPSS (LSFL) and their homogeneity as a function of single pulse fluence and effective fluence dose in the range from 0.067 to 1.070 J cm −2 and from 50 to 1280 J cm −2 , respectively. Here, a 1064 nm laser with a pulse duration of 13 ps was utilized to create LIPSS on Ti6Al4V alloy. The morphology of the formed structures was analyzed using the two‐dimensional fast Fourier transform method applied to scanning electron microscopy images. Homogeneous LSFL with spatial periods are obtained for effective fluence doses of 50 and 160 J cm −2 . However, an increase in the effective fluence dose up to 640 J cm −2 led to the formation of so‐called large LIPSS and the disappearance of homogeneous LSFL. The evolution of LIPSS that has been observed is attributed to changes in the ratio and absolute values of the ablative and heating components of the laser pulse energy at different single and effective fluence doses. Our study contributes to the development of tailored surfaces with periodic structures on Ti alloys.

This study leverages x‐ray phase‐contrast tomography (XPCT) for detailed analysis of neurodegenerative diseases, focusing on the three‐dimensional (3D) visualization and quantification of neuropathological features within fixed human postmortem tissue. XPCT with synchrotron radiation offers micrometer and even sub‐micron resolution, enabling us to examine intra‐ and extraneuronal aggregates and inclusions such as Lewy bodies (LBs), granulovacuolar degeneration (GvD), Hirano bodies (HBs), neurofibrillary tangles (NFTs), β‐amyloid plaques, and vascular amyloid deposits in three dimensions. In the reconstructions, we identified the highest electron densities in Hirano and LBs, while NFTs exhibited no significant increase in XPCT contrast. Using cutting‐edge high‐resolution x‐ray synchrotron beamlines, we were now able to detect even detect subcellular differences in electron densities found in GvD. Small‐scale inhomogeneities of the electron density were also detected in LBs, potentially relating to inclusions of organelles. Additionally, we reveal here a peculiar 3D geometry of HBs and demonstrate the co‐occurrence with GvD in the same neuron. These findings underscore the potential of XPCT as a powerful, label‐free tool for spatially resolved neuropathological investigations, opening new avenues for the systematic 3D characterization of inclusions and aggregates in neurodegeneration.

BackgroundParkinson’s disease (PD) affects millions of people worldwide, but only 5–10% of patients suffer from a monogenic forms of the disease with Mendelian inheritance. SNCA, the gene encoding for the protein alpha-synuclein (aSyn), was the first to be associated with familial forms of PD and, since then, several missense variants and multiplications of the gene have been established as rare causes of autosomal dominant forms of PD. In this study, we report the identification of a novel SNCA mutation in a patient that presented with a complex neurogenerative disorder, and unconventional neuropathological findings. We also performed in depth molecular studies of the effects of the novel aSyn mutation.MethodsA patient carrying the novel aSyn missense mutation and the family members were studied. We present the clinical features, genetic testing—whole exome sequencing (WES), and neuropathological findings. The functional consequences of this aSyn variant were extensively investigated using biochemical, biophysical, and cellular assays.ResultsThe patient exhibited a complex neurodegenerative disease that included generalized myocloni, bradykinesia, dystonia of the left arm and apraxia. WES identified a novel heterozygous SNCA variant (cDNA 40G > A; protein G14R). Neuropathological examination showed extensive atypical aSyn pathology with frontotemporal lobar degeneration (FTLD)-type distribution and nigral degeneration pattern with abundant ring-like neuronal inclusions, and few oligodendroglial inclusions. Sanger sequencing confirmed the SNCA variant in one healthy, 86-year-old parent of the patient suggesting incomplete penetrance. NMR studies suggest that the G14R mutation induces a local structural alteration in aSyn, and lower thioflavin T binding in in vitro fibrillization assays. Interestingly, the G14R aSyn fibers display different fibrillar morphologies than Lewy bodies as revealed by cryo-electron microscopy. Cellular studies of the G14R variant revealed increased inclusion formation, enhanced membrane association, and impaired dynamic reversibility of serine‐129 phosphorylation.ConclusionsThe atypical neuropathological features observed, which are reminiscent of those observed for the G51D aSyn variant, suggest a causal role of the SNCA variant with a distinct clinical and pathological phenotype, which is further supported by the properties of the mutant aSyn.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00889-y.

Optogenetic control is used to manipulate the activity of specific cell types in vivo for a variety of biological and clinical applications. Here we report ChReef, an improved variant of the channelrhodopsin ChRmine. ChReef offers minimal photocurrent desensitization, a unitary conductance of 80 fS and closing kinetics of 30 ms, which together enable reliable optogenetic control of cells at low light levels with good temporal fidelity and sustained stimulation. We demonstrate efficient and reliable red-light pacing and depolarization block of ChReef-expressing cardiomyocyte clusters. We used adeno-associated-virus-based gene transfer to express ChReef in retinal ganglion cells, where it restores visual function in blind mice with light sources as weak as an iPad screen. Toward optogenetic hearing restoration, ChReef enables stimulation of the auditory pathway in rodents and non-human primates with nanojoule thresholds, enabling efficient and frequency-specific stimulation by LED-based optical cochlear implants.

Background: Heart failure (HF) is associated with an increased risk of cognitive impairment and hippocampal dysfunction, yet the underlying molecular mechanisms remain poorly understood. This study aims to investigate the role of microRNA (miRNA) networks in hippocampus-dependent memory recovery in a mouse model of HF. Methods: CaMKIIδC transgenic (TG) mice, a model for HF, were used to assess hippocampal function at 3 and 6 months of age. Memory performance was evaluated using hippocampus-dependent behavioral tasks. Small RNA sequencing was performed to analyze hippocampal miRNA expression profiles across both time points. Bioinformatic analyses identified miRNAs that potentially regulate genes previously implicated in HF-induced cognitive impairment. Results: We have previously shown that at 3 months of age, CaMKIIδC TG mice exhibited significant memory deficits associated with dysregulated hippocampal gene expression. In this study, we showed that these impairments, memory impairment and hippocampal gene expression, were no longer detectable at 6 months, despite persistent cardiac dysfunction. However, small RNA sequencing revealed a dynamic shift in hippocampal miRNA expression, identifying 27 miRNAs as “compensatory miRs” that targeted 73% of the transcripts dysregulated at 3 months but reinstated by 6 months. Notably, miR-181a-5p emerged as a central regulatory hub, with its downregulation coinciding with restored memory function. Conclusions: These findings suggest that miRNA networks contribute to the restoration of hippocampal function in HF despite continued cardiac pathology and provide an important compensatory mechanism towards memory impairment. A better understanding of these compensatory miRNA mechanisms may provide novel therapeutic targets for managing HF-related cognitive dysfunction.

ABSTRACTBackgroundOptogenetics is a cutting‐edge approach that can enable direct stimulation of gastric smooth muscle cells (SMC) by combining cell‐specific overexpression of light‐sensitive proteins with light stimulation. We previously demonstrated that direct optogenetic stimulation of gastric SMC via depolarization can restore contractility and food propulsion and could become a new treatment strategy for gastroparesis. The human receptor Neuropsin (hOPN5) enables activation of Gq signaling with UV light. Herein, we explore this new strategy for direct optogenetic stimulation of gastric SMC.MethodsWe used a transgenic mouse model expressing hOPN5 in fusion with eYFP. Antral longitudinal smooth muscle strips were used for isometric force measurements and whole stomachs for intragastric pressure measurements, comparing light stimulation to other stimuli. Adeno‐associated virus (AAV) serotypes were screened for efficiency in transducing cultured gastric SMC, and transduced cells were tested by Ca2+ imaging.ResultshOPN5 expression was restricted to and found in ~1/3 of SMC in the stomach. UV light induced isometric force and increased intragastric pressure only in transgenic mice similarly to electrical field stimulation and reached approximately 1/3 of the force induced by global depolarization and muscarinic receptor activation. Importantly, optical stimulation remained effective in an ex vivo gastroparesis model. AAV 2.5 was by far the most effective serotype for SMC transduction, and UV light triggered Ca2+ transients in SMC expressing hOPN5.ConclusionhOPN5 is a new and effective tool to directly stimulate gastric SMC to control contractility with light. Thus, it is an additional and complementary approach to light‐induced membrane depolarization to restore gastric motility.

Abstract Importance Numerous studies indicate that the traditional categorical classification of severe mental disorders (SMD), such as schizophrenia, bipolar disorders, and major depressive disorders, does not align with the underlying biology of those disorders as they frequently overlap in terms of symptoms and risk factors. Objective This study aimed to identify transdiagnostic patient clusters based on disease severity and explore the underlying biological mechanisms independently of the traditional categorical classification. Design We utilized data from 443 participants diagnosed with SMD of the PsyCourse Study, a longitudinal study with deep phenotyping across up to four visits. We performed longitudinal clustering to group patients based on symptom trajectories and cognitive performance. The resulting clusters were compared on cross-sectional variables, including independent measures of severity as well as polygenic risk scores, serum protein quantification, miRNA expression, and DNA methylation. Results We identified two distinct clusters of patients that exhibited marked differences in illness severity but did not differ significantly in age, sex, or diagnostic proportions. We found 19 serum proteins significantly dysregulated between the two clusters. Functional enrichment pointed to a convergence of immune system dysregulation and neurodevelopmental processes. Conclusion The observed differences in serum protein expression suggest that disease severity is associated with the convergence of immune system dysregulation and neurodevelopmental alterations, particularly involving pathways related to inflammation and brain plasticity. The identification of pro-inflammatory proteins among the differentially expressed markers underscores the potential role of systemic inflammation in the pathophysiology of SMD. These results highlight the importance of considering illness severity as a core dimension in psychiatric research and clinical practice and suggest that targeting immune-related mechanisms may offer promising new therapeutic avenues for patients with SMD. Key points Question Can analyzing symptom trajectories and cognitive profiles across diagnostic categories reveal clinically relevant subgroups in severe mental disorders? Findings In this longitudinal study of 443 individuals with severe mental disorders, two distinct clusters emerged, differing significantly in illness severity, with the more severe group displaying elevated pro-inflammatory serum proteins, suggesting an association between disease severity and inflammation. Meaning These findings suggest that transdiagnostic clustering clarifies shared mechanisms, underscores the importance of inflammation in severe mental disorders, and highlights a promising avenue for novel therapeutic approaches.

Aggregation intermediates play a pivotal role in the assembly of amyloid fibrils, which are central to the pathogenesis of neurodegenerative diseases. The structures of filamentous intermediates and mature fibrils are now efficiently determined by single-particle cryo-electron microscopy. By contrast, smaller pre-fibrillar α-Synuclein (αS) oligomers, crucial for initiating amyloidogenesis, remain largely uncharacterized. We report an atomic-resolution structural characterization of a toxic pre-fibrillar aggregation intermediate (I1) on pathway to the formation of lipidic fibrils, which incorporate lipid molecules on protofilament surfaces during fibril growth on membranes. Super-resolution microscopy reveals a tetrameric state, providing insights into the early oligomeric assembly. Time resolved nuclear magnetic resonance (NMR) measurements uncover a structural reorganization essential for the transition of I1 to mature lipidic L2 fibrils. The reorganization involves the transformation of anti-parallel β-strands during the pre-fibrillar I1 state into a β-arc characteristic of amyloid fibrils. This structural reconfiguration occurs in a conserved structural kernel shared by a vast number of αS-fibril polymorphs including extracted fibrils from Parkinson’s and Lewy Body Dementia patients. Consistent with reports of anti-parallel β-strands being a defining feature of toxic αS pre-fibrillar intermediates, I1 impacts viability of neuroblasts and disrupts cell membranes, resulting in an increased calcium influx. Our results integrate the occurrence of anti-parallel β-strands as salient features of toxic oligomers with their significant role in the amyloid fibril assembly pathway. These structural insights have implications for the development of therapies and biomarkers.