Pasupuleti, Raghavendra Rao

Abstract Background Mono-allelic Dehydrodolichyl Diphosphate Synthase ( DHDDS) variants are associated with juvenile Parkinsonism, developmental delay and seizures. Symptoms are progressive, and various mechanisms, such as defective glycosylation, lysosomal dysfunction and cholesterol accumulation have been hypothesized to underlie disease symptoms. There is no treatment for DHDDS-related disease. Methods Patient-derived cortical forebrain organoids were created to elucidate disease mechanisms and evaluate potential treatments. In these neuronal models, glycosylation, lipidomics, proteomics, cholesterol/ganglioside accumulation, mitochondrial function and electrophysiological activity were assessed. Finally, we investigated the effects of nicotinamide mononucleotide (NMN), identified through a yeast-based drug screen, in neuronal cell models and in six patients in an off-label, N-of-1, observational series. Results DHDDS-patient derived organoids showed visual signs of degeneration after four months of culturing. This was accompanied by significant cholesterol accumulation in astrocytes, decreased mitochondrial respiration and loss of deep-layer neurons. In addition, we identified glycosylation abnormalities, showing for the first time that glycosylation in human tissue is affected by monoallelic DHDDS variants. Proteomic analysis revealed altered protein expression of proteins involved in lipid metabolism, cytoskeletal organization and neuronal development. We found that oral Nicotinamide Mononucleotide supplementation led to significant improvement in mitochondrial respiration and electrophysiological parameters in organoids, concurring with clinical improvements in all of the treated patients, particularly regarding their ataxia and tremor. Conclusion Our findings reveal a progressive phenotype in DHDDS-patient-derived brain organoids, with mitochondrial dysfunction and astrocyte-specific metabolic alterations contributing to disease pathology. Notably, NMN treatment led to clinical improvements in patients with heterozygous DHDDS variants, highlighting its potential as a therapeutic strategy.

Abstract Introduction: The intricate cellular structure is a constantly evolving environment where cells interact, communicate, and adapt to their surroundings. The fate, role, and actions of each cell are shaped by its specific location within the tissue. Therefore, studying the spatial proteome is crucial for gaining a deeper understanding of physiological or pathological processes. With significant advancements in microscopy and mass spectrometry, it has become feasible to explore the cellular proteome at low or even single cell numbers. However, spatial proteomics is an evolving field with scope for newer sample preparation and data analysis techniques. Our objective was to develop a sample preparation workflow for analyzing proteome from limited cells extracted from formalin fixed paraffin embedded (FFPE) tissue using laser capture microdissection (LCM). Methods: One hundred cells from the FFPE normal colon tissue were extracted by LCM and collected into an AFA-compatible 96-well plate. Decrosslinking was performed at 900C for 50 minutes followed by lysis in a buffer composed of 100 mM triethylammonium bicarbonate and 0.1% n-dodecyl-β-D-maltoside using adaptive focused acoustic (AFA) energetics in scanning mode for 5 minutes. After lysis, the proteins were digested using Trypsin/Lys-C mix using AFA energetics for 1 hour. The resulting peptides were acidified with trifluoroacetic acid and analyzed on a timsTOF Ultra 2 mass spectrometer coupled to a nanoElute 2 liquid chromatography system using a 15 cm (75 μm) IonOpticks column, in DDA-PASEF mode for a total run time of 34 minutes. The data were searched against the UniProt Human Reviewed protein database using MSFragger. Results: We utilized AFA energetics to extract and digest proteins from a limited number of cells isolated from FFPE tissue using LCM. This experiment was conducted with technical replicates to ensure accuracy and reliability. A total of 3,723 proteins were identified across the three replicates, with replicate 1 identifying 3,895 proteins, replicate 2 identifying 3,754 proteins, and replicate 3 identifying 3,519 proteins. In total, we identified 21,005 peptides, with replicate 1 identifying 22,896 peptides, replicate 2 identifying 22,377 peptides, and replicate 3 identifying 17,741 peptides from 100 colon cells. Notably, we observed a 70% overlap in the proteins identified across the three replicates, which highlights the reproducibility of the approach. Conclusion: The entire sample preparation process, after cell collection, was completed within a concise four-hour timeframe, demonstrating the efficiency of this methodology for analyzing protein profiles from small cell populations. Further, this approach is highly adaptable to automation and high-throughput applications. Its adaptability and scalability make it a promising strategy for spatial proteomics applications. Citation Format: Dong-Gi Mun, Kiran Mangalaparthi, Daigo Gunji, Amy J. French, Raghavendra Pasupuleti, Cristine Charlesworth, Sameer Vasantgadkar, DEB BHATTACHARYYA, Akhilesh Pandey. An AFA assisted workflow for mass-spectrometry based proteomics of FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7657.