Urushitani, M. | bioRxiv Science

bioRxiv2026

Intervention-consistent causal-source recovery from covariance-response geometry reveals upstream organisation in sporadic ALS

Sporadic amyotrophic lateral sclerosis (sALS) lacks longitudinal molecular measurements, making it difficult to distinguish early disease-organising changes from downstream consequences. We present a training-free framework that extracts directional structure from static single-nucleus RNA-seq by applying discrete Hodge decomposition to gene co-expression dynamics across pseudotime-ordered donor states. The framework separates irreversible co-expression cascades from circular feedback structure and regresses out the component explained by the healthy co-expression network, allowing disease-specific organisation to be examined in isolation. Perturbation benchmarks show that experimentally imposed sources are recoverable from control-normalised off-diagonal covariance-response fields, whereas marginal variance and diagonal covariance controls do not recover the source. Applied to sALS primary motor cortex (24 donors, 10 cell types), the framework identifies oligodendrocytes as the most structurally upstream cell type and upper-motor-neuron-containing layers as the most structurally downstream (Oligo cell-type{varphi} = 0.900, with glial cell types preserving the healthy co-expression network topology, whereas neuronal cell types show collapse-dominant deformation). Cytoplasmic translation is the only pathway with reproducible cross-cell-type upstream enrichment. At the gene level, the ribosome-associated quality-control factor NEMF -- which appends C-terminal alanine-threonine tags ("CATylation") to nascent chains on stalled ribosomes -- shows disease-specific loss of co-expression coherence in seven of ten cell types despite essentially unchanged mRNA expression; the disease signal is decoupling from collision-response partners (GCN2, PKR), not expression-level change. Cross-cohort validation across three BA4 motor cortex cohorts (including two external cohorts; total N=107) reproduced the oligodendrocyte-upstream / upper-motor-neuron-downstream structural architecture (Oligo-preserved / ET-sink) in all three cohorts, with NEMF co-expression coherence loss replicated in two of three cohorts. These data support a brain-side, circuit-distal structural model in which oligodendrocyte-lineage stress occupies an upstream-like preserved compartment, while upper-motor-neuron-containing excitatory populations form a downstream sink. The pattern is consistent with -- but does not directly establish -- a cascade architecture in which oligodendrocyte stress structurally precedes motor neuron TDP-43 pathology, and would produce a clinical phenotype resembling dying-back (the conventional view of ALS, in which motor neuron pathology appears to begin at distal axons and spread retrogradely toward the cell body) yet originating centrally and glially. NEMF/CATylation network disruption is identified as a candidate intermediate structural node bridging oligodendrocyte stress and motor neuron TDP-43 pathology.

neuroscience↗

bioRxiv2022

Spinal cord extracts from sporadic ALS induce and propagate TDP-43 pathology in cerebral organoids

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.

neuroscience↗