Spatiotemporal transcriptomics reveals distinct responses of ALDH1A1-positive and ALDH1A1-negative midbrain dopaminergic neurons to alpha-synuclein overexpression

Stepek, C.-J.; Ryan, E. B.; Villegas-Salmeron, J.; New, F.; Chavda, K.; Henshall, D. C.; DiMonte, D. A.; Prehn, J. H. M.; Ulusoy, A.; Connolly, N. M. C.

doi:10.64898/2026.07.17.739121

bioRxiv · 10.64898/2026.07.17.739121

Spatiotemporal transcriptomics reveals distinct responses of ALDH1A1-positive and ALDH1A1-negative midbrain dopaminergic neurons to alpha-synuclein overexpression

Stepek, C.-J.·Ryan, E. B.·Villegas-Salmeron, J.·New, F.·Chavda, K.·Henshall, D. C.·DiMonte, D. A.·Prehn, J. H. M.·Ulusoy, A.·Connolly, N. M. C.

View original source ↗Export metadata ↓

Abstract

Parkinsons disease is characterized by the progressive and preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, and intraneuronal alpha-synuclein (Syn) accumulation. Dopaminergic neurons (DANs) are anatomically and molecularly heterogeneous, but the impact of Syn pathology on distinct subpopulations is not well defined. One midbrain DAN sub-population expresses Aldehyde Dehydrogenase 1A1 (ALDH1A1), an enzyme that detoxifies aldehyde by-products of dopamine metabolism, and has been associated with differential vulnerability. Here, we applied GeoMx spatial transcriptomics to profile ALDH1A1-positive (ALDH1A1+) and ALDH1A1-negative (ALDH1A1-) DAN subpopulations in the mouse midbrain in situ at 3- and 8-weeks following adeno-associated virus (AAV)-mediated Syn overexpression. Analyzing 10,532 genes, we identified robust transcriptional differences between ALDH1A1+ and ALDH1A1- DANs under control conditions, supporting their characterization as distinct molecular subpopulations. In AAV-Syn-injected mice, we observed increased Snca expression and a reduction in ALDH1A1- DANs in the ipsilateral substantia nigra. Syn overexpression induced subpopulation-specific and time-dependent transcriptional responses, with dysregulation in ALDH1A1- DANs characterized by early down-regulation of pathways related to synaptic function, neurotransmitter handling, and bioenergetics, including glycolysis. In contrast, ALDH1A1+ DANs displayed later up-regulation of genes enriched for Acetyl-CoA and cholesterol metabolism pathways, reflecting subpopulation-specific adaptations to Syn overexpression. Analysis of human single nucleus RNA-sequencing data revealed partial conservation of the metabolic dysregulation signature. Together, our findings show that murine midbrain ALDH1A1+ and ALDH1A1- DANs represent molecularly distinct subpopulations with divergent temporal responses to Syn overexpression, emphasizing the importance of cell-type and disease-stage context in studies of Parkinsons disease mechanisms.

Explore related subjects

neuroscience Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Stepek, C.-J., Ryan, E. B., Villegas-Salmeron, J., New, F., Chavda, K., Henshall, D. C., DiMonte, D. A., Prehn, J. H. M., Ulusoy, A., Connolly, N. M. C.. 2026-07-21. Spatiotemporal transcriptomics reveals distinct responses of ALDH1A1-positive and ALDH1A1-negative midbrain dopaminergic neurons to alpha-synuclein overexpression. https://doi.org/10.64898/2026.07.17.739121

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Neurodegeneration-inducing macromolecules exit the brain via nanovascular conduits formed by reticular fibroblasts

Accumulation of proteins such as amyloid beta (Abeta), hyperphosphorylated tau and alpha-synuclein within the brain alters neural information processing and causes neurodegeneration(1-3), but how toxic solutes are cleared from the brain remains highly controversial(4,5). Proposed exit routes include efflux across endothelial cells into the blood(6,7), and movement to the pial surface via vasomotion-induced pumping along spaces within arteriolar smooth muscle(8) or via outflow along the perivascular space of ascending venules promoted by water flux through astrocytes (the glymphatic system(9)). From the pial surface of the brain, drainage may continue to dural lymphatics, along the outer sheaths of exiting cranial nerves and across the cribriform plate(10-14). We now report the presence, in mice and humans, of 2 micron diameter conduits that remove fluorescently labelled tau and Abeta from the brain. These conduits form a spatially-organised mesh within the walls of penetrating arterioles and pial arteries, and around the surface of ascending venules and deep cerebral and pial veins. They course through the pial and arachnoid layers to span the CSF space, wrapping the brain and cranial nerves. They are formed of reticular fibroblasts, which label for VE-cadherin(15) and PDGFRalpha(16), the lymphatic markers(17) podoplanin, VEGFR3 and Prox1, and reticular fibroblast extracellular matrix components collagen I and VI(16,18-20). Parenchymal tau drains from the brain at a similar rate via arteriolar conduits and via conduits around venules, arguing against preferential removal by a glymphatic mechanism. In Alzheimer's disease model mice, Abeta is seen traversing these lymph node-like conduits. Modulation of molecular transfer via this route may accelerate or delay cognitive decline, and slowed transfer from arteriolar to pial-arachnoid conduits may initiate cerebral amyloid angiopathy.

neuroscience↗

bioRxiv2026

Analysis of the influence of gradual changes in matrix sentence similarity on neural envelope tracking

Neural tracking of speech is a well-established phenomenon in neuroscience. However, for speech signals with a fixed structure, significant correlations between speech envelopes and neurophysiological representations occur even for unheard sentences. We exploit a structured speech-in-noise matrix hearing test (Oldenburger Sentence Test, OLSA) to systematically quantify the relationship between acoustic sentence similarity and neural tracking. Simultaneous magnetoencephalography (MEG) and 76-channel electroencephalography (EEG) data, including 16 channels positioned directly around the ears (ear-EEG), were recorded from 21 young adults with normal hearing during the presentation of clean-speech audiobooks and OLSA sentences at six signal-to-noise ratios. A linear decoder trained on audiobooks reconstructed OLSA sentence envelopes. Reconstruction accuracies were compared using a linear mixed model across heard (matched) and unheard (mismatched) sentences of varying acoustic similarity. Significant reconstruction accuracies were achieved across MEG, EEG, and ear-EEG for both matched and mismatched sentences. For mismatched sentences, these accuracies gradually increased with their acoustic similarity to the heard speech data. The high similarity between sentences, which is especially prominent in matrix tests, can cause significant spurious tracking for mismatched stimuli. This effect can reach levels comparable to those of matched sentences and can be mistaken for true neural tracking. Robust neural tracking across modalities further supported the established viability of ear-EEG compared to whole-head systems.

neuroscience↗

bioRxiv2026

Seizures and tauopathy following neurotrauma are mediated by prion protein and metabotropic glutamate receptor 5

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

neuroscience↗