AraC binds the p75NTR transmembrane domain to induce neurodegeneration in mature neurons

BackgroundCytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer including acute myeloid leukaemia. However, after high dose AraC chemotherapy regime, patients develop severe neurotoxicity and neurodegeneration in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells, however, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesized that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukemia patients undergoing AraC treatment.MethodsTo determine the role of AraC-p75NTR signalling in degeneration of mature cerebellar granule neurons, we used primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurodegeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the direct interaction between AraC and p75NTR, we performed isothermal dose response-cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging.ResultsWe show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, AraC binds to Proline 252 and Cysteine 256 of the p75NTR transmembrane domain and selectively uncouples p75NTR from the NFκB survival pathway. This in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR.ConclusionOur findings identify p75NTR as a novel molecular target to develop treatments to counteract AraC-mediated neurodegeneration.

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

Genetic Mechanism for the Cyclostome Cerebellar Neurons Reveals Early Evolution of the Vertebrate Cerebellum

The vertebrate cerebellum arises at the dorsal part of rhombomere 1, induced by signals from the isthmic organizer. Two major cerebellar neuronal subtypes, granule cells (excitatory) and Purkinje cells (inhibitory), are generated from the anterior rhombic lip and the ventricular zone, respectively. This regionalization and the way it develops are shared in all extant jawed vertebrates (gnathostomes). However, very little is known about early evolution of the cerebellum. The lamprey, an extant jawless vertebrate lineage or cyclostome, possesses an undifferentiated, plate-like cerebellum, whereas the hagfish, another cyclostome lineage, is thought to lack a cerebellum proper. In this study, we found that hagfish Atoh1 and Wnt1 genes are co-expressed in the rhombic lip, and Ptf1a is expressed ventrally to them, confirming the existence of r1’s rhombic lip and the ventricular zone in cyclostomes. In later stages, lamprey Atoh1 is downregulated in the posterior r1, in which the NeuroD increases, similar to the differentiation process of cerebellar granule cells in gnathostomes. Also, a continuous Atoh1-positive domain in the rostral r1 is reminiscent of the primordium of valvula cerebelli of ray-finned fishes. Lastly, we detected a GAD-positive domain adjacent to the Ptf1a-positive ventricular zone in lampreys, suggesting that the Ptf1a-positive cells differentiate into some GABAergic inhibitory neurons such as Purkinje and other inhibitory neurons like in gnathostomes. Altogether, we conclude that the ancestral genetic programs for the formation of a distinct cerebellum were established in the last common ancestor of vertebrates.

Single-cell spatial transcriptomic analysis reveals common and divergent features of developing postnatal granule cerebellar cells and medulloblastoma

Background Cerebellar neurogenesis involves the generation of large numbers of cerebellar granule neurons (GNs) throughout development of the cerebellum, a process that involves tight regulation of proliferation and differentiation of granule neuron progenitors (GNPs). A number of transcriptional regulators, including Math1, and the signaling molecules Wnt and Shh have been shown to have important roles in GNP proliferation and differentiation, and deregulation of granule cell development has been reported to be associated with the pathogenesis of medulloblastoma. While the progenitor/differentiation states of cerebellar granule cells have been broadly investigated, a more detailed association between developmental differentiation programs and spatial gene expression patterns, and how these lead to differential generation of distinct types of medulloblastoma remains poorly understood. Here, we provide a comparative single-cell spatial transcriptomics analysis to better understand the similarities and differences between developing granule and medulloblastoma cells. Results To acquire an enhanced understanding of the precise cellular states of developing cerebellar granule cells, we performed single-cell RNA sequencing of 24,919 murine cerebellar cells from granule neuron-specific reporter mice (Math1-GFP; Dcx-DsRed mice). Our single-cell analysis revealed that there are four major states of developing cerebellar granule cells, including two subsets of granule progenitors and two subsets of differentiating/differentiated granule neurons. Further spatial transcriptomics technology enabled visualization of their spatial locations in cerebellum. In addition, we performed single-cell RNA sequencing of 18,372 cells from Patched+/− mutant mice and found that the transformed granule cells in medulloblastoma closely resembled developing granule neurons of varying differentiation states. However, transformed granule neuron progenitors in medulloblastoma exhibit noticeably less tendency to differentiate compared with cells in normal development. Conclusion In sum, our study revealed the cellular and spatial organization of the detailed states of cerebellar granule cells and provided direct evidence for the similarities and discrepancies between normal cerebellar development and tumorigenesis.