Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders. Key features • Compartmentalized human induced pluripotent stem cell (iPSC)-derived motor neuron-myotube co-cultures for modeling distal axonal pathology. • Microfluidic separation of somatic and distal axonal compartments enabling spatial perturbation and analysis. • Quantitative imaging of neurofilament heavy chain (NFH)-associated axonal degeneration and pTDP-43 accumulation. • Semi-automated workflow for a reproducible, scalable, and modular pipeline for image quantification.

Stroke is a leading cause of disability worldwide, yet effective treatments to regenerate damaged tissue remain elusive. We investigated tissue regeneration and functional recovery post-stroke in an animal model with a combinatorial approach leveraging human induced pluripotent stem cell-derived neural progenitor cells (NPCs) and a sustained-release formulation of a re-designed, thermostable chondroitinase ABC (ChASE37). We optimized an in situ gelling, injectable hyaluronic acid (HA)-based hydrogel with the incorporation of laminin to deliver NPCs to the cavity in a rat model of sub-acute ischemic stroke. We delivered ChASE37 by affinity release from a methylcellulose hydrogel (ChASE37-AR) to the stroke-injured brain by epicortical injection. ChASE37-AR retained long-term enzymatic activity, as demonstrated by degradation of inhibitory chondroitin sulfate proteoglycans (CSPGs) in the injured tissue, with no evidence of immunogenicity. Each treatment - ChASE37-AR, NPCs, and their co-delivery - significantly improved motor function as early as 3 weeks after a single administration. Critically, only the combined therapy supported long-term survival and neuronal differentiation of transplanted cells, suggesting that ChASE37-AR has a role beyond CSPG degradation. This strategy presents a modular, clinically relevant platform for tissue regeneration post-stroke. Together, these findings suggest that the co-delivery of NPCs and sustained-release ChASE37 may be promising for stroke treatment.

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons (MNs) differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12147-8.