The small GTPases RhoA and Rac1 regulate cerebellar development by controlling cell morphogenesis, migration and foliation

AbstractThe small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology

Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3-KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

RhoA-ROCK Signaling as a Therapeutic Target in Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBIs, which range in severity from mild to severe, occur when a traumatic event, such as a fall, a traffic accident, or a blow, causes the brain to move rapidly within the skull, resulting in damage. Long-term consequences of TBI can include motor and cognitive deficits and emotional disturbances that result in a reduced quality of life and work productivity. Recovery from TBI can be challenging due to a lack of effective treatment options for repairing TBI-induced neural damage and alleviating functional impairments. Central nervous system (CNS) injury and disease are known to induce the activation of the small GTPase RhoA and its downstream effector Rho kinase (ROCK). Activation of this signaling pathway promotes cell death and the retraction and loss of neural processes and synapses, which mediate information flow and storage in the brain. Thus, inhibiting RhoA-ROCK signaling has emerged as a promising approach for treating CNS disorders. In this review, we discuss targeting the RhoA-ROCK pathway as a therapeutic strategy for treating TBI and summarize the recent advances in the development of RhoA-ROCK inhibitors

The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly

<div><p>The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.</p></div

Abnormal morphology and incorrect innervation in the spinal cord of RhoA cKO mice.

<p>(A) Dark field photos demonstrated that the dorsal funiculi of RhoA cKO mutant mice at the cervical spinal cord were more shallow and widened compared to the control and heterozygous mice. RhoA cKO mice exhibited a remarkable increase in the amount of gray matter at the midline. The width (horizontal line in A, left panel) and the height (vertical line in A, left panel) of funiculi were measured. Scale bar: 500 µm. (B) The ratio of width to height measurements for the dorsal funiculus of RhoA cKO mice was significantly greater than that of control and heterozygous mice (p<0.001). No significant differences were observed between control and heterozygous mice (p>0.05) (C). At the midline, the space between the dorsal column and ventral column of gray matter of RhoA cKO mice was significantly greater compared to control and heterozygous mice (p<0.001). n = 3–4 mice per group. Data are represented as the mean ± SD. *** = <i>p</i><0.001 (ANOVA followed by the Student-Newman-Keuls test). (D) Schematic drawing illustrating the strategy used for labeling spinal interneurons. Crystals of rhodamine dextran were applied unilaterally to control and RhoA cKO isolated mouse spinal cords at L4 (red rectangle). Contralateral projections were then visualized by imaging the labeled spinal cords at L2 (black dashed box) with an epifluorescence microscope. (E) Many spinal interneuron axons aberrantly cross the midline at L2 in the spinal cords of RhoA cKO mice, but not control littermates. Lower panels show an enlarged image of the area boxed in the upper panels. Scale bar represents 200 µm. Three sections per animal and N = 4 animals were analyzed per genotype.</p

Generation and characterization of mice containing a conditional allele of<i>RhoA.</i>

<p>(A) Schematic diagram of the strategy used to generate RhoA conditional knockout mice. A conditional <i>RhoA</i> allele was created by inserting two LoxP sites in a region of the <i>RhoA</i> gene flanking exon 3 and part of intron 3 (<i>RhoA^fl/fl</i>). An internal Frt-flanked neomycin (Neo) casette was also introduced as a selection marker, which was subsequently removed by crossing mutant mice with mice expressing Flippase (Flp recombination). The region of the <i>RhoA</i> gene between the two loxP sites was then excised in neuroprogenitor cells by crossing the <i>RhoA^fl/fl</i> mice with Nestin-Cre mice (Cre recombination). (B) PCR genotyping of <i>RhoA^fl/fl</i> (control), <i>RhoA^fl/+;Nestin-Cre</i> (RhoA het), and <i>RhoA^fl/fl;Nestin-Cre</i> (RhoA cKO) mice. Top gel, <i>RhoA</i> allele; bottom gel, <i>Cre</i>. (C) Protein isolated from the brains of adult control, RhoA het, and RhoA cKO mice were immunoblotted with antibodies against RhoA or GAPDH (loading control) to assess loss of RhoA expression.</p

Loss of RhoA inhibits ephrinB3-induced growth cone collapse and disrupts ephrinB3 midline expression.

<p>(A) Representative images of growth cones from control and RhoA cKO cortical neurons after a 30 min incubation with preclustered ephrinB3-Fc or Fc alone. Scale bar:10 µm. (B) Quantification of the growth cone collapse response of control and RhoA cKO neurons treated with ephrinB3-Fc or Fc (*p<0.001, ANOVA followed by the Tukey test). (C) Cortical neurons from RhoA cKO mice express EphA4 at levels similar to control neurons. (D) Percentage of ephrinB3-induced growth cone collapse in rat cortical neurons pretreated or not with the ROCK inhibitor Y-27632 (*p<0.001, ANOVA followed by the Tukey test). (E) Expression of ephrinB3 is lost from the midline at L2 in RhoA cKO mice. Arrowheads show ephrinB3 positive staining in the spinal cord of P5 mice. Scale bar: 10 µm. Three sections per animal and N = 3 animals were analyzed per genotype.</p

RhoA cKO mice display a rabbit-like hopping gait.

<p>(A) Control littermates display alternate limb movements, while RhoA cKO mice show a highly abnormal synchronous gait. Arrowheads indicate the position of hindlimbs with respect to each other. (B) Gait analysis of control and RhoA cKO mice. Non-toxic paint was applied to the forepaws (red) and hindpaws (black) of mice, and then they were allowed to walk on a piece of white paper to record the placement pattern of their footprints. (C) Determination of the distance between the right and left paw (a) and the distance between the same paw (b) to assess the degree of parallel movement of the limbs (comparison of a/b ratios). Data are represented as the mean ± S.E.M. (N = 6 animals per genotype), *p<0.001 (Student’s t-test).</p