Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development
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This work investigates the cellular and cerebellar origins of trisomy 21 (Down syndrome) phenotypes. One human chromosome 21 gene is Pericentrin (PCNT), encoding a component of the centrosome. The authors use several models with 3 or 4 copies of human chromosome 21 (or mouse equivalents) to reveal how increasing PCNT gene dosage alters ciliogenesis and ciliary signaling.
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Abstract
Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.
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Author Response
Reviewer #3 (Public Review):
The PCNT gene is found on human chromosome 21, and the same group previously showed that its increased expression is associated with reduced trafficking to the centrosome and reduced cilia frequency, which suggests a possible connection between cilia and ciliary trafficking, SHH signaling, and Down syndrome phenotypes. Jewett et al build upon this prior work by closely examining the trafficking phenotypes in cellular models with different HSA21 ploidy, or its mouse equivalent, thereby increasing the copy number of PCNT (3 or 4 copies of HSA21). They show that most of the trafficking defects can be reversed through the knockdown of PCNT in the context of HSA21 polyploidy. They also begin to examine the in vivo consequences of these trafficking disruptions, using a mouse model (Dp10) that …
Author Response
Reviewer #3 (Public Review):
The PCNT gene is found on human chromosome 21, and the same group previously showed that its increased expression is associated with reduced trafficking to the centrosome and reduced cilia frequency, which suggests a possible connection between cilia and ciliary trafficking, SHH signaling, and Down syndrome phenotypes. Jewett et al build upon this prior work by closely examining the trafficking phenotypes in cellular models with different HSA21 ploidy, or its mouse equivalent, thereby increasing the copy number of PCNT (3 or 4 copies of HSA21). They show that most of the trafficking defects can be reversed through the knockdown of PCNT in the context of HSA21 polyploidy. They also begin to examine the in vivo consequences of these trafficking disruptions, using a mouse model (Dp10) that partially recapitulates trisomy 21, including an increased copy number of PCNT. While I think this work advances our understanding of the trafficking defects caused by increased PCNT and has significant implications for our understanding of the cellular basis of a major hereditary human disorder, some improvements can be made to strengthen the conclusions and improve readability.
Major points:
I'm a little confused by the authors' conclusion that the increased PCNT levels in T21 and Q21 result in delayed but not attenuated ciliogenesis. The data show lower percentages of ciliated cells at all time points analyzed (Fig 1E) by quite a large margin in both T21 and Q21. Do the frequencies of cilia in the T21 or Q21 cells ever reach the same level as D21, say after 48-72 hours? If not it seems like not simply a delay. A bit more clarity about this point is needed.
We have now performed a ciliation time course in RPE1 D21, T21, and Q21 cells over 7 days. Our new data confirms that increasing HSA21 dosage delays but does not abolish ciliogenesis (Fig S1H). By day 3 of serum depletion, D21 and T21 cells reach similar ciliation frequencies, and after 4 days all three cell lines reach similar ciliation frequencies.
The in vivo analysis of the cerebellum was interesting and important but it felt a bit incomplete given that it was a tie between the cell biology and a specific DS- associated phenotype. For example, it is interesting that the EGL of the P4 Dp10 pups is thinner. Does this translate into noticeable defects in cerebellar morphology later? Is there a reduction in proliferation that follows the reduced cilia frequency? I think it would be possible to look at the proliferation and cerebellar morphology at some additional stages without becoming an overly burdensome set of experiments. At a minimum, are there defects in cerebellar morphology at P21 or in the adult mice? The authors allude to developmental delays in these animals - maybe that complicates the analysis? But additional exploration and/or discussion on this point would help the paper.
We have now analyzed P21 animals and found no significant differences in ciliation frequency or gross cerebellar morphology at this age. This is consistent with our new tissue culture data demonstrating that HSA21 ploidy delays but does not abolish ciliogenesis. We cannot rule out long term changes in neuronal processes or glial cells, but we believe this analysis is outside the scope of this paper.
It was a bit unclear to me why specific cell lines were used to model trisomy 21 and why this changed part way through the paper. I understand the justification for making the Dp10 mice- to enable the in vivo analysis of the cerebellum, but some additional rationale for why the RPE cell line is initially used and then the switch back to mouse cells would improve readability.
The rationale for switching to MEFs was twofold. First, Shh ciliary signaling cannot be easily studied in RPE1 cells. Therefore, ciliary function via Smoothened localization or GLI1 transcription, needed to be performed in a different cell line and the most commonly used line is MEFs. Second, the Dp mice allowed us to tease apart contributions to cilia defects from separate regions of HSA21. We have worked to clarify this point in the text.
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eLife assessment
This work investigates the cellular and cerebellar origins of trisomy 21 (Down syndrome) phenotypes. One human chromosome 21 gene is Pericentrin (PCNT), encoding a component of the centrosome. The authors use several models with 3 or 4 copies of human chromosome 21 (or mouse equivalents) to reveal how increasing PCNT gene dosage alters ciliogenesis and ciliary signaling.
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Reviewer #1 (Public Review):
This group previously demonstrated that trisomy 21 causes an increase in PCNT levels, and this increase leads to pericentrosomal crowding and inhibition of ciliogenesis in fibroblasts. The authors here use trisomy and tetrasomy 21 retinal pigment epithelium cells generated by microcell-mediated chromosome transfer (MMCT) and previously generated mouse models of human trisomy 21. The well-quantified data and well-reasoned paper compellingly demonstrate that modestly increased PCNT levels can attenuate ciliogenesis and may result in trisomy 21-associated phenotypes such as cerebellar growth defects.
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Reviewer #2 (Public Review):
This manuscript builds on previous work from the Pearson lab showing that one aspect of the trisomy 21 phenotype could be caused by an increase in the amount of pericentrin (PCNT), a component of the centrosome. The earlier work showed that the increase in PCNT is sufficient to reduce the frequency of ciliation in trisomy 21 cells and that the increased PCNT is often in the form of protein aggregates along microtubules proximal to the centrosome (in a preprint). Here they use several models with 3 or 4 copies of human chromosome 21 or the mouse equivalent to examine the defect in cilium formation at the level of specific proteins and the signaling function of the cilium. This is a substantial contribution that furthers the evidence for the authors' favored model of excess PCNT causing some form of …
Reviewer #2 (Public Review):
This manuscript builds on previous work from the Pearson lab showing that one aspect of the trisomy 21 phenotype could be caused by an increase in the amount of pericentrin (PCNT), a component of the centrosome. The earlier work showed that the increase in PCNT is sufficient to reduce the frequency of ciliation in trisomy 21 cells and that the increased PCNT is often in the form of protein aggregates along microtubules proximal to the centrosome (in a preprint). Here they use several models with 3 or 4 copies of human chromosome 21 or the mouse equivalent to examine the defect in cilium formation at the level of specific proteins and the signaling function of the cilium. This is a substantial contribution that furthers the evidence for the authors' favored model of excess PCNT causing some form of pericentrosomal crowding that hinders the ability of other molecules, complexes, and/or vesicles to get to the right place at the right time. The work makes excellent use of cell lines and mice previously generated for the study of trisomy 21, making for well-controlled experiments in a situation where this is particularly important (only 1.5 x increased expression). One does wish that it were possible to do the PCNT depletion in more of the experiments than the single one shown, but that is understandable given the amount of work required and the uncertainty associated with RNAi depletion.
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Reviewer #3 (Public Review):
The PCNT gene is found on human chromosome 21, and the same group previously showed that its increased expression is associated with reduced trafficking to the centrosome and reduced cilia frequency, which suggests a possible connection between cilia and ciliary trafficking, SHH signaling, and Down syndrome phenotypes. Jewett et al build upon this prior work by closely examining the trafficking phenotypes in cellular models with different HSA21 ploidy, or its mouse equivalent, thereby increasing the copy number of PCNT (3 or 4 copies of HSA21). They show that most of the trafficking defects can be reversed through the knockdown of PCNT in the context of HSA21 polyploidy. They also begin to examine the in vivo consequences of these trafficking disruptions, using a mouse model (Dp10) that partially …
Reviewer #3 (Public Review):
The PCNT gene is found on human chromosome 21, and the same group previously showed that its increased expression is associated with reduced trafficking to the centrosome and reduced cilia frequency, which suggests a possible connection between cilia and ciliary trafficking, SHH signaling, and Down syndrome phenotypes. Jewett et al build upon this prior work by closely examining the trafficking phenotypes in cellular models with different HSA21 ploidy, or its mouse equivalent, thereby increasing the copy number of PCNT (3 or 4 copies of HSA21). They show that most of the trafficking defects can be reversed through the knockdown of PCNT in the context of HSA21 polyploidy. They also begin to examine the in vivo consequences of these trafficking disruptions, using a mouse model (Dp10) that partially recapitulates trisomy 21, including an increased copy number of PCNT. While I think this work advances our understanding of the trafficking defects caused by increased PCNT and has significant implications for our understanding of the cellular basis of a major hereditary human disorder, some improvements can be made to strengthen the conclusions and improve readability.
Major points:
I'm a little confused by the authors' conclusion that the increased PCNT levels in T21 and Q21 result in delayed but not attenuated ciliogenesis. The data show lower percentages of ciliated cells at all time points analyzed (Fig 1E) by quite a large margin in both T21 and Q21. Do the frequencies of cilia in the T21 or Q21 cells ever reach the same level as D21, say after 48-72 hours? If not it seems like not simply a delay. A bit more clarity about this point is needed.
The in vivo analysis of the cerebellum was interesting and important but it felt a bit incomplete given that it was a tie between the cell biology and a specific DS-associated phenotype. For example, it is interesting that the EGL of the P4 Dp10 pups is thinner. Does this translate into noticeable defects in cerebellar morphology later? Is there a reduction in proliferation that follows the reduced cilia frequency? I think it would be possible to look at the proliferation and cerebellar morphology at some additional stages without becoming an overly burdensome set of experiments. At a minimum, are there defects in cerebellar morphology at P21 or in the adult mice? The authors allude to developmental delays in these animals - maybe that complicates the analysis? But additional exploration and/or discussion on this point would help the paper.
It was a bit unclear to me why specific cell lines were used to model trisomy 21 and why this changed part way through the paper. I understand the justification for making the Dp10 mice- to enable the in vivo analysis of the cerebellum, but some additional rationale for why the RPE cell line is initially used and then the switch back to mouse cells would improve readability.
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