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When the obvious fails, look at the unexpected: interneuron individual behavior affects the population migration.

Posted by , on 19 March 2018

The story behind our paper: Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis. Carla G. Silva, Elise Peyre, Mohit H. Adhikari, Sylvia Tielens, Sebastian Tanco, Petra Van Damme, Lorenza Magno, Nathalie Krusy, Gulistan Agirman, Maria M. Magiera, Nicoletta Kessaris, Brigitte Malgrange, Annie Andrieux, Carsten Janke, Laurent Nguyen

 

The research behind this article is a good example of how looking beyond expected results can lead to unexpected discoveries.  This story is not what we originally thought it was going to be about. But simplistic hypotheses failed to explain standard measurements and this lead us to a deeper question.

 

In the beginning:

In the 1970’s a Purkinje cell degeneration mutant mouse had been studied1 and the deficient gene responsible for the phenotype was later shown to be Ccp12. This gene codes for an enzyme, called carboxypeptidase 1, capable of digesting chained glutamates on tubulin (the building block of microtubules) or on a wide range of additional proteins3.

When we first started working in the lab of Laurent Nguyen, we were given access to the CCP1 conditional knock out mouse3. At the time, the lab was focusing on understanding the mechanisms underlying interneuron migration during cortical development. This migration is a fascinating process. The cortical interneurons are born in regions located far away from the residence of their future, mature selves. As the embryonic brain is forming, a steady flow of interneurons moves away from their birth place to reach the cortical plate. The developing brain environment gives cues to steer them in the right direction, both repulsing them from the zones they should not invade and attracting them towards correct paths. Each individual interneuron senses its environmental cues and adapts its movements accordingly. This information is translated to directed movements thanks to the dynamic remodelling of the cytoskeleton, which gives scaffolding structure to the cell but also generates contraction and pulling forces4. Microtubules and acto-myosin fibres are the main components of the interneuron cytoskeleton and their contractions and dynamics are tightly regulated. Two general levels of cytoskeletal regulation can be found: 1) gene-encoded signalling cascades regulate contraction or polymerisation/depolymerisation; 2) posttranslational modifications of cytoskeleton components can fine-tune the movement. One of these posttranslational modifications is the addition/removal of glutamate.

We reasoned that by removing Ccp1 and hyperglutamylating cytoskeletal proteins, we would subtly modify interneuron migration without stalling cells.

 

The phenotype observed:

Our primary observation was that too many interneurons were invading the cortex upon loss of Ccp1 expression during development. It would then take us about 2 years to understand why.

We first characterised cortical invasion by cortical interneuron in more detail. We showed that between E12.5 and birth, more Ccp1 cKO interneurons were invading the cortex of mouse embryos. This was an unusual phenotype since so far, cytoskeletal modifications lead to delayed migration and reduced number of interneurons in the cortex.

In parallel we characterized the cellular pattern of migration of these interneurons, using time lapse video microscopy. We were able to follow their behaviour as they moved toward their destinations.

 

GFP-expressing cortical interneurons migrating away from MGE explants prepared from E13.5 CCP1 WT embryos. Duration of recording is 5h.

 

Not only could we measure the average speed of displacement but we were also able to study the way interneurons cortical interneurons were moving. We observed that indeed, the behaviour of interneurons lacking Ccp1 was slightly changed despite having a preserved average speed of migration. Instead of alternating phases of nuclear pauses and large amplitude jumps called nucleokinesis5, mutant interneurons were pausing for a shorter amount of time and the amplitude of their nucleokinesis was reduced. Smaller jumps compensating shorter pauses, amounting to similar average speed.

The next question was to understand what could explain our observation of larger number of interneurons in the cortical plate?

 

The simplistic hypotheses:

To answer this question we tested a large number of hypotheses that yielded negative results and frustrating statistically non-significant histograms.

We first tested if more cortical interneurons were generated in upon loss of Ccp1. Our genetic recombination tool (Dlx5.6 CRE mouse line) was expressed in a small portion of dividing cells. We counted the number of mitotic cells, the number of cells replicating their DNA and the general cell cycle phases distribution of our CCP1 mutant cells and nothing was changed in our mutant.

We then reasoned that if more cortical interneurons were reaching the cortex, it could be explained by an increased survival, leading to fewer cells dying on the way. However, we did not detect any reduction of apoptosis in Ccp1 cKO cortical interneurons as compare to their controls.

Another plausible explanation was that mutant cortical interneurons would take “short-cut” pathways to the cortex and thus would be more efficiently reaching their destination. We counted the number of cortical interneurons crossing the striatum, a no-go zone for cortical interneurons and again, we did not measure any differences.

Finally, we tested whether loss of Ccp1 could affect cell fate and favour generation of cortical interneurons at the expense of oligodendrocyte that are born in overlapping regions of the forebrain from a common pool of progenitors. We showed that the mutant brain did not lack oligodendrocytes. To be sure that the conditional knockout of Ccp1 knock-down in cortical interneurons was not resulting in higher interneurons generation through a non-cell autonomous activity, we counted the number of recombinant cells in the whole brain using FACS. At E13.5 we measured the same total number of cells in Ccp1 cortical interneurons brains as compared to WT controls. This observation suggested that more cortical interneurons were displaced in the cortical compartment in Ccp1 cKO embryos.

None of the above hypotheses proved to be true. So why were there more cells invading the cortex?

 

The unexpected:

By this point, the project was 3 years old and although several hypotheses had been eliminated we were not closer to an explanation for our phenotype. We sat down and looked at the data. It was the moment to decide to either put the project aside or take a fresh look at the whole thing in a different light. What if the slight change of individual behaviour could influence the way the entire population moved? To help us with this new hypothesis we called upon our colleague Mohit Adhikari, a talented physicist, to help modelling the behaviour of our cells in silico. Using the parameters of speed and pause duration of migrating cortical interneurons measured by time lapse experiments, we generated trials of surrogate cells displacements. In this in silico experiment, the interneurons are challenged along the simplest path, a straight line on a 2D plane to isolate and test the effect of kinetic behaviour on the population displacement.

 

Displacement of cell surrogates with WT or Ccp1 cortical interneuron migration parameters. Displacement of all cell surrogates in both groups (Group A gray and Group B red); lighter marker shade indicates higher total displacement of a surrogate. Horizontal bars mark the highest displacement thresholds crossed by 75% of surrogates in each group. Duration of simulation is 600min.

 

The simulation allowed us to conclude that a difference in the kinetic properties of migration, such as shorter pauses and smaller jumps, increases recruitment at short distances from the starting point of CCP1 mutant interneurons in the developing cortex. We finally had a possible explanation for our phenotype, and the reason why had been staring at us from the start.

 

The molecular regulation:

In parallel to running in silico simulations of cortical interneurons displacement, we set out to understand the molecular regulation of migration linked with the Ccp1 mutation. We first found that acto-myosin fibres, a component of the cytoskeleton, were not contracting normally in upon loss of Ccp1 in cortical interneurons. This observation was done in time lapse video microscopy experiments when cortical interneurons were electroporated with a acto-myosin contraction fluorescent probe.

 

Migration of pLifeAct-Ruby electroporated E13.5 WT or cKO Ccp1 cortical interneuron homochronic mixed cortical feeder

 

Mutant cortical interneurons were generating almost permanent actomyosin-derived forces instead of pausing. These forces were, however, either not strong or not focused enough at the rear of the nucleus to grant correct nuclear jump. We looked at the phosphorylation state of the myosin light chain (MLC), the effector of acto-myosin contraction, and we observed hyper phosphorylation, confirming MLC over-activation. We then turned our attention to the kinase that phosphorylates MLC, MLCK3,6, and showed that it was not only a substrate of our enzyme Ccp1 in cortical interneurons but also that it was hyperactive in the mutant cortical interneurons. This showed a new molecular regulation of acto-myosin contraction and helped us understanding what lead to the abnormal movement behaviour of our mutant interneurons.

 

The cherry on the cake:

Finally our last question was: why is cortical invasion by cortical interneurons regulated during cerebral cortical development? We raised this question because we had noticed that the mutant brains were able to correct their number of cortical interneurons after birth by killing off the supplementary cells by apoptosis. If the problem can be corrected later why does cortical interneurons cortical invasion need to be regulated? To answer this question we looked at the cortex, the compartment receiving migrating interneurons. During our birth dating experiments, we injected BrdU to birthdate and track cohorts of cortical interneurons. We analysed BrdU injected brains at E13.5 and noticed an abnormal proliferation of the projection neuron progenitors, the intermediate progenitors in the cortex of Ccp1 mutant brains. This lead to an overproduction of projection neurons persisting in postnatal brains. This result was unexpected as our genetic recombination tool is specific to subpallial regions and does not target the cortical progenitor cell populations. Interestingly this increased proliferation was detected in regions with higher numbers of interneurons. We postulated that during development, the number of interneurons entering the cortex regulates the production of the projection neurons with which they will form connections later on. To test this hypothesis we analysed another mouse model (Nkx2.1 mutant). In these mutants brains no cortical interneurons entered the cortex and this resulted in a decreased proliferation of intermediate progenitors, suggesting again a regulatory link between the number of interneurons in the cortex and the proliferation of intermediate progenitors.

These observations are original and they unravel a regulatory crosstalk between cortical progenitors and interneurons regulating corticogenesis progression. This could be relevant for human diseases as increased cortical thickness are described in patients suffering from autism spectrum disorder 7,8.

 

Conclusion

Overall this work took 5 years to complete and in the end, it is far from what we expected it to be when we first started. The challenge of getting to an explanation beyond the obvious lead us to unexpected and novel results.

 

By Elise Peyre and Carla Silva

 

1 Mullen, R. J., Eicher, E. M. & Sidman, R. L. Purkinje cell degeneration, a new neurological mutation in the mouse. Proceedings of the National Academy of Sciences of the United States of America 73, 208-212 (1976).

2 Harris, A. et al. Regenerating motor neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases. Molecular and cellular neurosciences 16, 578-596, doi:10.1006/mcne.2000.0900 (2000).

3 Rogowski, K. et al. A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 143, 564-578, doi:10.1016/j.cell.2010.10.014 (2010).

4 Peyre, E., Silva, C. G. & Nguyen, L. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex. Frontiers in cellular neuroscience 9, 129, doi:10.3389/fncel.2015.00129 (2015).

5 Bellion, A., Baudoin, J. P., Alvarez, C., Bornens, M. & Metin, C. Nucleokinesis in tangentially migrating neurons comprises two alternating phases: forward migration of the Golgi/centrosome associated with centrosome splitting and myosin contraction at the rear. The Journal of neuroscience : the official journal of the Society for Neuroscience 25, 5691-5699, doi:10.1523/JNEUROSCI.1030-05.2005 (2005).

6 Somlyo, A. P. & Somlyo, A. V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiological reviews 83, 1325-1358, doi:10.1152/physrev.00023.2003 (2003).

7 Courchesne, E. et al. Neuron number and size in prefrontal cortex of children with autism. Jama 306, 2001-2010, doi:10.1001/jama.2011.1638 (2011).

8 Zielinski, B. A. et al. Longitudinal changes in cortical thickness in autism and typical development. Brain : a journal of neurology 137, 1799-1812, doi:10.1093/brain/awu083 (2014).




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