From Cradle to Grave: Control of Purkinje Cell Dendrite Morphology By a Single Transcription Factor RORα
Material below summarizes the article RORα Regulates Multiple Aspects of Dendrite Development in Cerebelar Purkinje Cells In Vivo, published on September 9, 2015, in JNeurosci and authored by Yukari H. Takeo, Wataru Kakegawa, Eriko Miura, and Michisuke Yuzaki.
In the nervous system, each neuron subtype develops unique dendritic architecture that widely differs in the number of branches and the 3D orientation. Since excitatory synaptic inputs are formed mostly on dendrites, proper formation and maintenance of the dendritic architecture is fundamental for neural circuit function. Indeed, abnormal dendritic morphology is reported in brains of several neuropsychiatric disorders as well as neurodegenerative diseases. However, the mechanism of how neurons develop and maintain subtype-specific dendritic arbors is poorly understood.
Cerebellar Purkinje cells (PCs) have one of the most elaborate and distinctive dendritic trees and have served as a useful model for research of dendritogenesis. They typically have a single primary stem dendrite, from which abundant dendritic branches arise and spread out flatly in a single plane. At birth, post-migratory PCs show a spindle shape with long primitive dendrites and are scattered in the cerebellar cortex. This phase is called “fusiform” stage. By four days after birth, the cell bodies of PCs line in a single cell layer. At this phase, the long dendrites are retracted and PCs exhibit compact “stellate cell” shape with numerous short dendrites orienting radially from cell body. Then, by around postnatal day eight, short dendrites disappear and single thick stem dendrites with several branches emerge. For the next two weeks, a highly branched dendritic tree grows in a plane and numerous dendritic spines are formed densely on the distal branches. Thus, in a few postnatal weeks, the dendrites of PCs maturate through multiple stages accompanied by an extension and retraction of dendrites.
Earlier studies using in vitro culture, spontaneous and X ray-induced mutant mice have revealed that maturation of PC dendrites is regulated by both genetic programs and extrinsic, environmental factors. A candidate molecule of intrinsic regulator of PC dendrites — retinoic acid-related orphan receptor α (RORα) — is a transcription factor expressed predominantly in PCs. RORα was originally identified as a gene responsible for a particular line of mutant mice (called staggerer mice) more than half a century ago. Staggerer mice, in which most PCs die during early postnatal weeks, show severe cerebellar ataxia. The remaining PCs have a strikingly atrophic shape similar to wild-type PCs at the fusiform stage, leading to a hypothesis that RORα is involved in dendrite maturation. Using an in vitro slice culture experiment, Boukhtouche et al. previously demonstrated that RORα is involved in early postnatal dendritic retraction during the transition from the fusiform stage to the “stellate cell” stage. More recently, Chen et al. reported that RORα is also required for maintenance of PC dendrites after the second week in vivo. Nevertheless, exact roles of RORα in dendrite development in vivo have remained elusive because some of the features of PC dendrites are not completely recapitulated in cultures. In addition, the effects caused by the disruption of RORα during the early developmental stages could be masked by dynamic dendritic growth and regression in the later stages.
In this study, to clarify RORα function in PCs at specific developmental stages in vivo, we combined the in utero electroporation method, which enabled PC-specific transfection as early as E11.5, with the drug-induced gene knockdown, knockout, or overexpression system. Using these tools, we have demonstrated that RORα plays essential roles, much wider than previously described, in different aspects of dendrite development throughout all stages of PC maturation.
First, we knocked down endogenous RORα in PCs in vivo from E11.5 when PCs have just finished terminal differentiation. As previously suggested, we found that RORα was required for monolayer formation and dendrite retraction during the transition from the fusiform stage to the “stellate-cell” stage between postnatal day (P) 0 and P4. Next, we asked whether RORα is involved in dendrite maturation after the “stellate-cell” stage. Using drug-induced temporally controlled knockdown or knockout methods, we revealed that RORα was necessary for the formation of single primary dendrites by eliminating perisomatic short dendrites after “stellate-cell” stage. In addition, RORα was necessary for formation and maintenance of dendritic branches to become full-fledged PCs even after stem dendrites were determined. These data for the first time unveiled roles for RORα during postnatal dendritic remodeling in vivo.
One of the prominent morphological features of PC dendrites is numerous spines on their distal branches. The spine formation of PCs is thought to be operated by an intrinsic program because spines are formed in PCs in the complete absence of presynaptic inputs in many mutant mice. We found that loss of RORα after the “stellate-cell” stage reduces spiny dendritic protrusions from PCs. These data suggest that RORα function is crucial for spine formation in PCs.
Interestingly, excess RORα levels have adverse effects on maturation of dendrites and spine formation. When exogenous RORα is overexpressed after “stellate-cell” stage, formation of mature dendrites was impaired. The number of dendritic spiny protrusions was also decreased. Instead, each spine’s head size was significantly enlarged. These results suggest that keeping moderate levels of RORα activity is important for balanced formation of dendrites and spines.
Finally, we found that RORα is essential for maintenance of dendritic morphology and synaptic functions in mature PCs. Knockdown or knockout of RORα in mature PCs at three postnatal weeks caused severe dendritic atrophy and reduction of excitatory postsynaptic responses. Those RORα-deficient PCs often had abnormal somatic or axonal swelling, which are the features of degenerating PCs. Intriguingly, decreased RORα expression in PCs of spinocerebellar ataxia type1 (SCA1) model mice is reported to determine disease severity in SCA1 model mice. Our results that RORα is required for survival of mature PCs are consistent with the SCA1 pathology.
Our study has revealed that RORα plays multifaceted roles in the formation and maintenance of PC dendrites during development and throughout life. These findings shed light on the mechanism by which a single transcriptional factor governs subtype-specific morphogenesis. Further studies are warranted to reveal each molecular mechanism at different maturation stages.
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RORα Regulates Multiple Aspects of Dendrite Development in Cerebellar Purkinje Cells In Vivo. Yukari H. Takeo, Wataru Kakegawa, Eriko Miura, Michisuke Yuzaki. The Journal of Neuroscience Sep 2015, 35(36): 12518-12534; DOI: 10.1523/JNEUROSCI.0075-15.2015
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