Advanced ERC grant „NeuroCentro“
The centrosome is a universal eukaryotic cell organelle that typically orchestrates the cytoskeleton and cell division. As this function is exerted across many cell types, not much attention has been paid to the differences of centrosomes between cells.
We encountered a protein, the originally called AT-Hook transcription factor AKNA, that was located with exquisite cell type specificity at the centrosome. It is expressed higher and located at the centrosome in differentiating neural stem cells, but not self-renewing neural stem cells (Camargo et al., Nature 2019). Indeed, Akna mediates the delamination of the differentiating stem cells from their neuroepithelial niche, by orchestrating microtubule organizing center activity at the centrosome and recruiting proteins that stabilize adherence junctions to the centrosome thereby weakening the junctional complexes. Beyond this specificity, we also observed that Akna is required for migration into the next layer, the subventricular zone, but then needs to be turned off for young neurons to migrate further and switch from a multipolar to bipolar state (Camargo et al. Nature 2019). This highly specific regulation of one protein at the centrosome prompted us to explore centrosome composition at proteome-wide scale to determine if Akna is the exception or the rule.
Aim 1 Identify novel cell type-specific centrosome functions
Within this project we performed the first total centrosome proteome analysis of human neural stem cells and neurons. This revealed a striking several hundred of proteins that had never been found at the centrosome before (O’Neill et al., Science 2022). Moreover, it revealed that 50% of the centrosome proteome is exchanged during differentiation from neural stem cells to neurons. As a further surprise we found as top GO term RNA binding and RNA processing proteins at the centrosome, including splicing factors, such as PRPF6.
Beyond this, we could also find a significant and specific overlap with specific disease variants. For example, some disease variants show significant overlap only with the neuronal centrosome proteome, and others, such as those of patients with periventricular heterotopia, only with the neural stem cell centrosome proteome. As these are variants only, i.e. mutations found in single patients, the centrosome proteome may allow prioritizing those that are likely functional. Indeed, we could verify this as well as elucidate the key question of how a ubiquitous protein could cause a brain-specific phenotype when mutated. This is the case for the splicing factor PRPF6 that we found at the centrosome specifically in neural cells and the mutation observed in a patients with periventricular heterotopia also caused some cells to remain at the ventricle in a preclinical model. The mutant protein caused aberrations in alternative splicing and one of its targets it a microtubule associated protein kinase. As we found the RNA of this also at the centrosome and could rescue the phenotype of the mutant PRPF6 protein by co-electroporating the correctly spliced form of this kinase, we suggest that PRPF6 localization at the centrosome matters as it brings some of its crucial splicing targets also to the centrosome.
Aim 2 Explore neuronal subtype-specific migration and centrosome function
Neuronal migration is known to be very diverse, with some neurons migrating along radial glial cells, others along axons, others without any apparent specific substrate and yet others in glial tubes as community. However, the molecular mechanisms involved in these specific modes of migration are not yet well understood.
One molecular candidate mediating different types of migration is Akna. In Camargo et al., Nature 2019 we have observed that Akna is required for the migration into the subventricular zone, but needs to be down-regulated for migration out of the subventricular zone. This means that the former requires centrosomal microtubule organizing center activity, while the later requires it to be absent.
Interestingly, Akna plays no role in another centrosome function, namely the early neuronal polarization and axon formation, but indeed for migration. This was shown recently in collaboration with Frank Bradke’s lab (Vinopal et al., Neuron 2023).
Amongst migrating neurons in different brain regions, we find Akna in neuroblasts of the rostral migratory stream. We have now generated conditional Akna knock-out mouse lines and use these to explore the function of Akna in migrating neuroblasts in this region. Subcellular analysis and live imaging is performed with Prof. Armen Saghatelyan, University of Laval, Quebec and University of Ottawa, Canada. For recent achievements of our collaborative work see Gengatharan et al., Cell 2021.
The centrosome is a universal eukaryotic cell organelle that typically orchestrates the cytoskeleton and cell division. As this function is exerted across many cell types, not much attention has been paid to the differences of centrosomes between cells.
We encountered a protein, the originally called AT-Hook transcription factor AKNA, that was located with exquisite cell type specificity at the centrosome. It is expressed higher and located at the centrosome in differentiating neural stem cells, but not self-renewing neural stem cells (Camargo et al., Nature 2019). Indeed, Akna mediates the delamination of the differentiating stem cells from their neuroepithelial niche, by orchestrating microtubule organizing center activity at the centrosome and recruiting proteins that stabilize adherence junctions to the centrosome thereby weakening the junctional complexes. Beyond this specificity, we also observed that Akna is required for migration into the next layer, the subventricular zone, but then needs to be turned off for young neurons to migrate further and switch from a multipolar to bipolar state (Camargo et al. Nature 2019). This highly specific regulation of one protein at the centrosome prompted us to explore centrosome composition at proteome-wide scale to determine if Akna is the exception or the rule.
Aim 1 Identify novel cell type-specific centrosome functions
Within this project we performed the first total centrosome proteome analysis of human neural stem cells and neurons. This revealed a striking several hundred of proteins that had never been found at the centrosome before (O’Neill et al., Science 2022). Moreover, it revealed that 50% of the centrosome proteome is exchanged during differentiation from neural stem cells to neurons. As a further surprise we found as top GO term RNA binding and RNA processing proteins at the centrosome, including splicing factors, such as PRPF6.
Beyond this, we could also find a significant and specific overlap with specific disease variants. For example, some disease variants show significant overlap only with the neuronal centrosome proteome, and others, such as those of patients with periventricular heterotopia, only with the neural stem cell centrosome proteome. As these are variants only, i.e. mutations found in single patients, the centrosome proteome may allow prioritizing those that are likely functional. Indeed, we could verify this as well as elucidate the key question of how a ubiquitous protein could cause a brain-specific phenotype when mutated. This is the case for the splicing factor PRPF6 that we found at the centrosome specifically in neural cells and the mutation observed in a patients with periventricular heterotopia also caused some cells to remain at the ventricle in a preclinical model. The mutant protein caused aberrations in alternative splicing and one of its targets it a microtubule associated protein kinase. As we found the RNA of this also at the centrosome and could rescue the phenotype of the mutant PRPF6 protein by co-electroporating the correctly spliced form of this kinase, we suggest that PRPF6 localization at the centrosome matters as it brings some of its crucial splicing targets also to the centrosome.
Aim 2 Explore neuronal subtype-specific migration and centrosome function
Neuronal migration is known to be very diverse, with some neurons migrating along radial glial cells, others along axons, others without any apparent specific substrate and yet others in glial tubes as community. However, the molecular mechanisms involved in these specific modes of migration are not yet well understood.
One molecular candidate mediating different types of migration is Akna. In Camargo et al., Nature 2019 we have observed that Akna is required for the migration into the subventricular zone, but needs to be down-regulated for migration out of the subventricular zone. This means that the former requires centrosomal microtubule organizing center activity, while the later requires it to be absent.
Interestingly, Akna plays no role in another centrosome function, namely the early neuronal polarization and axon formation, but indeed for migration. This was shown recently in collaboration with Frank Bradke’s lab (Vinopal et al., Neuron 2023).
Amongst migrating neurons in different brain regions, we find Akna in neuroblasts of the rostral migratory stream. We have now generated conditional Akna knock-out mouse lines and use these to explore the function of Akna in migrating neuroblasts in this region. Subcellular analysis and live imaging is performed with Prof. Armen Saghatelyan, University of Laval, Quebec and University of Ottawa, Canada. For recent achievements of our collaborative work see Gengatharan et al., Cell 2021.
