Serine 1283 in extracellular matrix glycoprotein Reelin is crucial for Reelin′s function in brain development

Deficiency in the extracellular matrix glycoprotein Reelin severely affects migration of neurons during development. The function of serine at position 1283 in Reelin has remained uncertain. To explore its relevance we generated rlnA/A mice that carry alanine instead of serine at position 1283, thereby disrupting the putative casein kinase 2 (CK2) phosphorylation site S1283DGD. Mutated mice displayed reeler-like locomotor behavior, abnormal brain anatomy and decrease of Reelin RNA and protein levels during development and in adulthood. Since serine 1283 was previously proposed to mediate proteolysis of adhesion molecules, we investigated proteolysis of cell adhesion molecule L1 and found it normal in rlnA/A mice. Neuronal migration in the embryonic rlnA/A cerebral cortex was impaired, but rescued by in utero electroporation of the Reelin fragment N-R6 containing the putative CK2 phosphorylation site. In rlnA/A mice migration of cerebellar granule cells in vitro was promoted by application of wild-type but not by mutated Reelin. In cerebellar neuron cultures, Reelin expression was decreased upon inhibition of ecto-phosphorylation by CK2. Biochemically purified wild-type, but not mutated Reelin was found phosphorylated. Altogether, the results indicate that ecto-phosphorylation at serine 1283 rather than proteolytic processing of adhesion molecules by Reelin plays an important role in Reelin functions.

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.

A cerebellar-prepontine circuit for tonic immobility triggered by inescapable threat

Sudden changes in the sensory environment are frequently perceived as threats and may provoke defensive behavioral states. One such state is tonic immobility, a conserved defensive strategy characterized by a powerful suppression of movement and motor reflexes. Tonic immobility has been associated with multiple brainstem regions and cell types, but the underlying circuit is not known. Here, we demonstrate that a strong vibratory stimulus evokes tonic immobility in larval zebrafish defined by suppression of exploratory locomotion and sensorimotor responses. Using a circuit-breaking screen and targeted neuron ablations we show that cerebellar granule cells and a cluster of glutamatergic ventral prepontine neurons (vPPNs) that express key stress-associated neuropeptides are critical components of the circuit that suppresses movement. The complete sensorimotor circuit transmits information from primary sensory neurons through the cerebellum to vPPNs to regulate reticulospinal premotor neurons. These results show that cerebellar regulation of a neuropeptide-rich prepontine structure governs a conserved and ancestral defensive behavior that is triggered by inescapable threat.

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.

Regulating synchronous oscillations of cerebellar granule cells by different types of inhibition

Synchronous oscillations in neural populations are considered being controlled by inhibitory neurons. In the granular layer of the cerebellum, two major types of cells are excitatory granular cells (GCs) and inhibitory Golgi cells (GoCs). GC spatiotemporal dynamics, as the output of the granular layer, is highly regulated by GoCs. However, there are various types of inhibition implemented by GoCs. With inputs from mossy fibers, GCs and GoCs are reciprocally connected to exhibit different network motifs of synaptic connections. From the view of GCs, feedforward inhibition is expressed as the direct input from GoCs excited by mossy fibers, whereas feedback inhibition is from GoCs via GCs themselves. In addition, there are abundant gap junctions between GoCs showing another form of inhibition. It remains unclear how these diverse copies of inhibition regulate neural population oscillation changes. Leveraging a computational model of the granular layer network, we addressed this question to examine the emergence and modulation of network oscillation using different types of inhibition. We show that at the network level, feedback inhibition is crucial to generate neural oscillation. When short-term plasticity was equipped on GoC-GC synapses, oscillations were largely diminished. Robust oscillations can only appear with additional gap junctions. Moreover, there was a substantial level of cross-frequency coupling in oscillation dynamics. Such a coupling was adjusted and strengthened by GoCs through feedback inhibition. Taken together, our results suggest that the cooperation of distinct types of GoC inhibition plays an essential role in regulating synchronous oscillations of the GC population. With GCs as the sole output of the granular network, their oscillation dynamics could potentially enhance the computational capability of downstream neurons.

The physiological role of Homer2a and its novel short isoform, Homer2e, in NMDA receptor-mediated apoptosis in cerebellar granule cells

Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.

The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

An experimental prediction: granule cells can perform linearly non-separable computations

Multiple studies show how dendrites might extend some neurons' computational capacity. These studies leave a large fraction of the nervous system unexplored. Here we demonstrate how a modest dendritic tree can allow cerebellar granule cells to implement linearly non-separable computations. Granule cells' dendrites do not spike and these cells' membrane voltage is isopotential. Conjunction of Boolean algebra and biophysical modelling enable us to make an experimental prediction. Granule cells can perform linearly non-separable computations. The standard neuron model used in the artificial network, aka the integrate and fire, cannot perform such type of computations. Confirming the prediction we provide in the present work would change how we understand the nervous system.