Trotti Research

Our laboratory – as an integral part of the Weinberg ALS Center - is dedicated to deciphering the molecular and cellular neurodegenerative mechanisms in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We leverage cutting-edge non-animal models, including induced pluripotent stem-derived neuronal and non-neuronal cells, 3D neural cultures, and mouse models, combined with advanced imaging and computational analysis. We aim to uncover novel therapeutic targets and biomarkers to pave the way for effective treatments for these neurodegenerative conditions.

Techniques. We integrate complementary cellular, molecular, in vivo, and systems-level approaches to define the mechanisms linking metabolic stress, aberrant RNA biology, and innate immune activation to neurodegeneration in C9orf72-ALS/FTD and related disorders. We use patient-derived iPSCs and isogenic controls differentiated into cortical and spinal motor neurons, astrocytes, microglia-like cells, and long-term cerebral organoids, together with rapid i3-neuron systems and CRISPR/Cas9 genome engineering for targeted knock-in/out and repeat correction, enabling precise interrogation of cell-autonomous and non–cell-autonomous mechanisms. Cellular phenotyping combines high-content imaging, live-cell microscopy, Seahorse mitochondrial stress testing (OCR/ECAR), ROS and redox assays, mitochondrial membrane potential measurements, ATP/NAD⁺ quantification, and automated quantification of TDP-43 mislocalization, dipeptide repeat aggregation, DNA damage, senescence, and innate immune activation. In parallel, we employ AAV-mediated gene delivery and multiple transgenic mouse models for behavioral testing, survival analyses, and multiplex histopathology. Mechanistically, we integrate Western blotting, qPCR/ddPCR, ELISA/MSD/SIMOA cytokine profiling, splicing/cryptic exon analyses, and RAN translation assays to interrogate translational control and RNA processing. These are coupled to multi-omics pipelines encompassing stable isotope (¹³C) metabolic flux analysis, targeted and untargeted LC–MS/MS metabolomics, bulk and single-nucleus RNA-seq, spatial transcriptomics, quantitative and spatial proteomics, and computational integration using dimensionality reduction, network analysis, and mixed-effects modeling. Finally, we translate discoveries into therapeutic strategies through small-molecule screening, drug-response profiling in organoids and mice, extracellular vesicle biomarker development, and immune-engineering approaches such as CAR-T–based microglial targeting, all conducted under standardized SOPs, blinded quantification, and rigorous quality control to ensure reproducibility and preclinical relevance.

In our studies of transposable elements and innate immunity, we investigate how endogenous retroelements function not only as sources of genomic instability but also as active modulators of neuroinflammatory signaling. We have delineated a previously unrecognized pathogenic axis in Alzheimer’s disease and ALS in which aberrant activation of HERVK and related retrotransposons promotes the accumulation of RNA–DNA hybrids and cytosolic nucleic acid species that engage cGAS–STING signaling, triggering sustained type I interferon responses and microglia-driven neuroinflammation. These findings establish transposable element dysregulation as a proximal driver of innate immune activation rather than a secondary consequence of degeneration. Our goal is to identify actionable nodes that can be therapeutically targeted to mitigate inflammatory toxicity in ALS and related neurodegenerative disorders.

We investigate the critical role EVs play in transmitting disease-associated factors in ALS. Our studies focus on how these vesicles contribute to neuroinflammation, particularly in the context of C9ORF72 mutations. We also explore the potential of EVs as therapeutic carriers, aiming to harness their properties to mitigate disease progression. By understanding the impact of EVs in ALS, we strive to uncover new therapeutic strategies that could offer relief to those affected by this debilitating condition.

We developed transdifferentiation-based human cell models to study how innate immune sensing pathways contribute to neurodegeneration in ALS/FTD. By directly converting patient fibroblasts into induced neurons and glia cells while preserving age-related and epigenetic signatures, we generate disease-relevant systems that more faithfully capture stress, mitochondrial dysfunction, and inflammatory phenotypes than conventional reprogrammed iPSCs. Using these models, we investigate the accumulation of mitochondrial double-stranded RNA and cytosolic nucleic acid species that activate RIG-I–like receptors and downstream interferon signaling. Combining live-cell imaging, metabolic and mitochondrial assays, RNA–DNA hybrid detection, and transcriptomic profiling, we dissect how mitochondrial stress engages RIG-I–mediated innate immunity to drive neuroinflammation and neuronal vulnerability. This platform enables mechanistic studies and rapid testing of therapeutic strategies aimed at dampening maladaptive immune activation in ALS/FTD.

At the ALS Center, our team of scientists specializing in Computational Biology, and Bioinformatics is dedicated to supporting data analysis and data visualization across a wide range of experiments and research studies. Leveraging cutting-edge methodologies, we develop robust data processing pipelines for single-cell and spatial multi-omics, as well as transcriptomic, proteomic, and complex imaging analyses. This integrated approach is crucial for identifying predictive biomarkers and uncovering novel therapeutic targets.