Alzheimer's disease (AD) is a heterogeneous neurodegenerative disorder, highlighting the need to identify novel molecular regulators for effective treatment development. Angiogenin (ANG), a stress-responsive ribonuclease that inhibits apoptosis by generating 5'-tRNA fragments, is a candidate whose expression and regulation in AD is not understood. Here, we investigated ANG expression and regulation using AD cell and animal models, postmortem human brain tissue, and transcriptomic datasets (n = 645). We found that ANG is dysregulated in AD in a sex-dependent manner, altering downstream levels of 5'-tiRNAGly-GCC. Our analysis revealed female-specific molecular subtypes, absent in males: Subtype 1 featured low ANG levels with increased inflammation and neuronal death; subtype 2 exhibited higher ANG expression and intermediate pathology; subtype 3, marked by the highest ANG levels, showed reduced inflammation, slower cognitive decline, and extended survival. These findings position ANG as a key modulator of neuroinflammation and apoptosis in AD, highlighting its potential as a treatment strategy.

tRNA-derived fragments have emerged as critical regulators in various biological processes, but reliable methods for their quantification remain a challenge due to their small size and extensive RNA modifications. In this study, we present the newly developed Complementary DNA Oligonucleotide Direct In-Gel Quantification (cDINGQ) method for tRF analysis and compare it with traditional radioactive [ 32 P] Northern blotting, non-radioactive approaches, and high-throughput Illumina sequencing under different experimental conditions. The cDINGQ method, utilizing Cy5-labeled hybridization probes, offers high specificity and sensitivity for detecting tRFs with significantly reduced processing time and costs. By applying these techniques to an Alzheimer’s disease (AD) cell model, we demonstrate the reliability of these methods in detecting subtle variations in tRF abundance. Our findings highlight the sensitivity, specificity, and applicability of each method, addressing limitations such as RNA input requirements and probe hybridization conditions. The study further explores the utility of these methods for detecting tRFs in various biological contexts, emphasizing their potential for future research and biomarker discovery in disease-related studies.

Nanopore sequencing preserves native DNA and RNA modifications and encodes them directly in electrical signal, but extracting this information requires accurate signal-to-sequence alignment. Existing tools perform this reliably yet often demand metadata handling or format conversion. We present Fishnet, a lightweight and fast aligner that reimplements the Remora alignment algorithm while removing surrounding overhead. Fishnet produces near-identical alignments more than thirty times faster and provides a simple command-line interface for alignment and downstream formatting. Benchmarks demonstrate high concordance between alignment tools and Fishnet’s mostly superior speed. Analyses of synthetic RNA constructs prove its practical utility for streamlined studies of modified nucleotides.

Abstract tRNA modifications are critical regulators of RNA stability, decoding fidelity, and cellular stress adaptation, yet their contribution to human neurodegenerative disease remains poorly understood. Beyond their established functions in translational control, emerging evidence shows that RNA modifications influence neurogenesis, neurodevelopment, neuronal function, brain-cell differentiation, and cellular plasticity. Consequently, dysregulation of these molecular processes is increasingly recognized as a mechanistic contributor to neurodegenerative disorders. Alzheimer’s disease (AD), characterized by amyloid pathology, synaptic dysfunction, and progressive neuronal loss, has recently been linked to disturbances in RNA metabolism, suggesting that alterations in the epitranscriptome may represent an underexplored dimension of AD pathophysiology. Here, we systematically profiled the tRNA epitranscriptome across cellular and animal models of AD, as well as in human postmortem brain tissue from non-demented controls and AD patients, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method enables highly sensitive quantification of RNA modifications, with limits of detection in the low femtomole range. Across our models, we identified a conserved yet sex-specific remodeling of the tRNA modification landscape in AD. Because therapeutic options and early diagnostic tools for AD remain limited, we leveraged these findings to develop a tRNA-centered RNA-modification score that integrates both nucleobase-specific modification patterns and neuropathological disease severity into a quantitative metric. Together, our findings identify the tRNA epitranscriptome as a unifying molecular sex-specific signature of AD, linking disease pathology and sex to impaired RNA metabolism. This line of research opens a new path toward establishing early biomarkers or diagnostic tools for AD. Graphical abstract

Nanopore technology offers real-time sequencing opportunities, providing rapid access to sequenced data and allowing researchers to manage the sequencing process efficiently, resulting in cost-effective strategies. Here, we present focused case studies demonstrating the versatility of real-time transcriptomics analysis in rapid quality control for long-read RNA-seq. We illustrate its utility through four experimental setups: (1) transcriptome profiling of distinct human cellular populations, (2) identification of experimentally enriched transcripts, (3) transcriptional analysis of cells under heat shock conditions, and (4) identification of experimentally manipulated genes (knockout and overexpression) in several yeast strains. We show how to perform multiple layers of quality control as soon as sequencing has started, addressing both the quality of the experimental and sequencing traits. Real-time quality control measures assess sample/condition variability and determine the number of identified genes per sample/condition. Furthermore, real-time differential gene/transcript expression analysis can be conducted at various time points post-sequencing initiation (PSI), revealing dynamic changes in gene/transcript expression between two conditions. Using real-time analysis, which occurs in parallel to the sequencing run, we identified differentially expressed genes/transcripts as early as 1 hr PSI. These changes were consistently observed throughout the entire sequencing process. We discuss the new possibilities offered by real-time data analysis, which have the potential to serve as a valuable tool for rapid and cost-effective quality checks in specific experimental settings and can be potentially integrated into clinical applications in the future.

Mitochondrial dysfunction is considered one of the key drivers of neurodegeneration and pathological aging, characterized by impaired energy production, oxidative stress, disrupted mitophagy, and biogenesis. Because mitochondria regulate bioenergetics, redox balance, and neuronal survival, therapeutic strategies that restore mitochondrial integrity are of growing interest. This review outlines mechanisms of mitochondrial function and failure, links them to Alzheimer’s and Parkinson’s disease, and summarizes evidence on phytochemicals and mitochondria-targeted small molecules, which enhance biogenesis, mitophagy, respiratory efficiency, and antioxidant defence in preclinical models together with life-style interventions. Although many compounds demonstrate preventive rather than restorative benefit and clinical evidence remains limited, next-generation approaches, including nanoparticles for mitochondrial delivery, mtDNA editing, and mitochondrial transfer, suggest increasing therapeutic potential. We underline that future success will rely on improved delivery, synergistic combinations, and rigorous clinical trials. Mitochondria-directed therapies may ultimately provide disease-modifying or preventive strategies for neurodegenerative disorders.

DNMT2 (TRDMT1) is a human RNA methyltransferase implicated in various disease processes. However, small-molecule targeting of DNMT2 remains challenging due to poor selectivity and low cellular availability of known S-adenosylhomocysteine (SAH)-derived ligands. In this study, a DNA-encoded library (DEL) screen identified five non-SAH-like chemotypes that selectively bind DNMT2, including three peptidomimetics. Orthogonal assays confirmed target engagement, and X-ray crystallography revealed a previously unknown allosteric binding pocket formed via active site loop rearrangement. Guided by structural insights, the authors optimized a lead compound with a K D of 3.04 μM that reduces m5C levels in MOLM-13 tRNA and synergizes with doxorubicin to impair cell viability. These inhibitors exhibit unprecedented selectivity over other methyltransferases, offering a promising scaffold for future DNMT2-targeting therapeutics. Beyond pharmacological implications, the study provides conceptual advances in understanding allosteric modulation and structural plasticity of DNMT2.

Methodological developments in biomedical research are currently moving towards single-cell approaches. This allows for a much better spatial and functional characterization of, for example, the deterioration of cells within a tissue in response to noxae. However, subcellular resolution is also essential to elucidate whether observed impairments are driven by an explicit organelle. Here, we use the Single Cellome™ System SS2000 (Yokogawa) to investigate the local effects of Aβ plaque-like deposits (characteristic for Alzheimer’s disease) on mitochondria in the mouse microglial cell line SIM-A9. First, the specificity of subcellular extraction is demonstrated by detecting subcellular staining and RT-qPCR concerning marker genes by comparing nuclear and mitochondrial samples. Oxygen consumption and gene expression is then assessed in cells near and far from peptide deposits. Mostly, all analyses confirm the high specificity and integrity of the sampled material. In addition, impact of the peptide deposits occur concerning spatial distribution of the cells: e.g., oxygen consumption is only reduced in cells close to Aβ deposits but not in proximity to deposits of biologically inactive Aβ (scrambled) or in far distance. Moreover, a distance-related gene expression pattern occurs, demonstrating the local initiation of mitochondrial changes of microglia when approaching toxic peptide deposits. Aspiration of cellular content accompanied by confocal microscope-usage within the SS2000 allows investigation of organelle-enriched fractions and sheds light on selective effects of Aβ peptides.

Mitochondria are the central organelles that allow eukaryotic cells to efficiently convert nutrients into energy for cellular functions such as anabolic reactions,movement,and regulation.A reduction in the number of mitochondria or the occurrence of dysfunctional mitochondria leads to serious diseases such as the Leigh syndrome.However,such changes have also been connected to Alzheimer's disease(AD)and many more diseases of different organ systems and occur during the aging process.

RNA modifications play a crucial role in various cellular functions. Here, we present ModiDeC, a deep-learning-based classifier able to identify and distinguish multiple RNA modifications (N6-methyladenosine, inosine, pseudouridine, 2′-O-methylguanosine, and N1-methyladenosine) using direct RNA sequencing. Alongside ModiDeC, we provide an extensive database of in vitro-transcribed and synthetic sequences generated with both the new RNA004 chemistry and the old RNA002 kit. We show that RNA modifications can be accurately recognized and distinguished across different sequence motifs using synthetic data as well as in HEK293T cells and human blood samples. ModiDeC comes with a graphical user interface that allows easy customization and adaptation to specific research questions, such as learning and classifying additional RNA modifications and further sequence motifs. The reproducibility across samples, together with the low rate of false positives, underscores the potential of ModiDeC as a powerful tool for advancing the analysis of epitranscriptomes and RNA modification.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m 1 A mRNA methylation is highly discussed due to methodological differences. However, one single m 1 A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m 1 A site might be involved in the pathophysiology of Alzheimer’s disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid β (Aβ). Aβ mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m 1 A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m 1 A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m 1 A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aβ-induced mitochondrial dysfunction.

Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues.

The synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. Here we report three novel findings suggesting a completely new role of SV2A. First, we demonstrate that SV2A is localized at the outer mitochondrial membrane (OMM) using confocal and super-resolution microscopy. Second, Inactivation of SV2A in our cell and animal models leads to fragmented mitochondria. In addition, SV2A also affects the basal autophagic flux as well as mitophagy. Third, using proteomics analysis we demonstrate that SV2A interacts with the fission factor DRP1 and the autophagy factor ATG9A. Using AlphaFold3 we provide a first glimpse of the molecular interaction between DRP1 and SV2A. Our findings demonstrate that SV2A is not only a vesicular protein but also a mitochondrial protein in the OMM with defined functions regulating mitochondrial morphology and autophagy. ### Competing Interest Statement The authors have declared no competing interest.

The 5' cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high-quality synthetic standards via a new route, involving P(III)-amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one-step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl-alcohols. Stable-isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC-MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences.

Selective modulation of TRPC6 ion channels is a promising therapeutic approach for neurodegenerative diseases and depression. A significant advancement showcases the selective activation of TRPC6 through metalated type-B PPAP, termed PPAP53. This success stems from PPAP53's 1,3-diketone motif facilitating metal coordination. PPAP53 is water-soluble and as potent as hyperforin, the gold standard in this field. In contrast to type-A, type-B PPAPs offer advantages such as gram-scale synthesis, easy derivatization, and long-term stability. Our investigations reveal PPAP53 selectively binding to the C-terminus of TRPC6. Although cryoelectron microscopy has resolved the majority of the TRPC6 structure, the binding site in the C-terminus remained unresolved. To address this issue, we employed state-of-the-art artificial-intelligence-based protein structure prediction algorithms to predict the missing region. Our computational results, validated against experimental data, indicate that PPAP53 binds to the 777LLKL780-region of the C-terminus, thus providing critical insights into the binding mechanism of PPAP53.

The existence of mitochondria in eukaryotic host cells as a remnant of former microbial organisms has been widely accepted, as has their fundamental role in several diseases and physiological aging. In recent years, it has become clear that the health, aging, and life span of multicellular hosts are also highly dependent on the still-residing microbiota, e.g., those within the intestinal system. Due to the common evolutionary origin of mitochondria and these microbial commensals, it is intriguing to investigate if there might be a crosstalk based on preserved common properties. In the light of rising knowledge on the gut–brain axis, such crosstalk might severely affect brain homeostasis in aging, as neuronal tissue has a high energy demand and low tolerance for according functional decline. In this review, we summarize what is known about the impact of both mitochondria and the microbiome on the host’s aging process and what is known about the aging of both entities. For a long time, bacteria were assumed to be immortal; however, recent evidence indicates their aging and similar observations have been made for mitochondria. Finally, we present pathways by which mitochondria are affected by microbiota and give information about therapeutic anti-aging approaches that are based on current knowledge.

The selective modulation of TRPC6 ion channels has emerged as a promising therapeutic approach for treating neuro-degenerative diseases and depression. Here, we present a significant advancement in this field by demonstrating the se-lective activation of TRPC6 using a metallated type-B PPAP, designated as PPAP53. The success of PPAP53 is attributed to the utilization of the 1,3-diketone motif present in PPAPs for metal coordination. The metallated PPAPs exhibit water solubility and equipotent activity compared to hyperforin, which is a natural product and considered the gold standard in the field. Notably, and in sharp contrast to type-A PPAPs, type-B PPAPs possess unique properties such as synthetic ac-cessibility in gram scale, facile derivatization, being thermally stable and stable against photochemical oxidation. Our detailed investigations reveal that PPAP53 selectively binds to the C-terminus of TRPC6. Although cryo electron micros-copy has resolved the majority of the TRPC6 structure, the binding site in the C-terminus remained unresolved. To ad-dress this issue, we employed state-of-the-art artificial intelligence-based protein structure prediction algorithms, includ-ing AlphaFold2, ColabFold, and trRosetta, to predict the missing C-terminus region. Our computational results, validated against experimental data, indicate that PPAP53 binds to the 777LLKL780-region of the C-terminus, thus providing critical insights into the binding mechanism of PPAP53 with TRPC6.

Zusammenfassung Hintergrund Im Rahmen der gesundheitsökonomischen Analyse wurde in einem Piggyback-Ansatz die Kosteneffektivität des am Point of Care Apotheke erbrachten Präventionsprogrammes der GLICEMIA 2.0-Studie evaluiert, das versuchte, die Teilnehmenden in der Interventionsgruppe zu einer verbesserten glykämischen Kontrolle bei Typ-2-Diabetes mit nachhaltiger Incentivierung von gesundheits-bewusstem Verhalten, therapeutischer Compliance und Adhärenz heranzuführen. Die Kontrollgruppe erhielt ein passives Medikationsmanagement und ein Monitoring zum Diabetes-Status. Methode Primärer Endpunkt der GLICEMIA 2.0-Studie war die Stabilisierung des HbA1c-Wertes. Zur gesundheitsökonomischen Auswertung wurden inkrementale Unterschiede in Output-Veränderungen untersucht, definiert als Differenz der Häufigkeitsverteilung der HbA1c-Werte zwischen den Gruppen im zeitlichen Verlauf. Als Kostenparameter wurden direkte Programmkosten und antizipierte indirekte Kosten der Inanspruchnahme ärztlicher Leistungen erfasst. Zur Validierung von Kosteneffektivitätsschwellen wurde über die Bildung von ICER-Werten ein Net-Monetary-Benefit-Ansatz herangezogen. Ergebnis Die Interventionsgruppe erreichte signifikant höhere Verbesserungen der HbA1c-Werte. Durch die vorgenommene Risikostratifizierung über den Anfangsmedian der HbA1c-Werte können Kosteneffektivitätspotenziale bei hohen HbA1c-Ausgangswertengezeigt werden. Aufgrund des Untersuchungszeitraums sind keine langfristigen Unterschiede in Inanspruchnahmen ärztlicher Leistungen ersichtlich. Schlussfolgerung Das GLICEMIA 2.0-Programm indiziert deutliche Effektivitätspotenziale besonders bei höheren Risikograden. Der Wirkeffekt scheint die Adhärenz der Interventions- im Vergleich zur Kontrollgruppe befördert zu haben. Eine Ermittlung der unmittelbaren Wirkungshebel der Gruppenintervention würde aufgrund des Charakters einer komplexen Intervention einen längeren Zeitraum benötigen. Aufgrund eines fehlenden Follow-up können über längerfristige Effekte nur eingeschränkte Aussagen getroffen werden.