While the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. Here, we developed a high-content pipeline, the Large-area Spatial Transcriptomic (LaST) map, which can quantify singlecell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid, and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in early postnatal cortex, mostly persisted in adult mouse and human cortex. Single cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson’s disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To “humanize” this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking/ER quality control and mRNA metabolism/translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control/function, metal ion transport, transcriptional regulation and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9, VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2 and EIF4G1/PARK18) were confirmed in patient iPS cell-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms, and may facilitate patient stratification for targeted therapy.

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