The hidden side of the UPR signalling pathway

Initially recognized as  homeostatic signalling pathway that helps cells to cope with cellular stress, the Unfolded Protein Response (UPR) has kept an unexpected activity well hidden up until now. It would also seem that it plays an important physiological role in the development of the nervous system. That's what Laurent Nguyen and his team reveal in an article published in Trends in Neurosciences.

Pregnancy and monitoring the foetus' development arouses feelings of both excitement and apprehension of future parents. Is it growing well? Is it still moving? Are its limbs well formed? Is its brain developing properly? In the majority of cases, the news is good and the baby is developing properly. Sometimes, parents are increasingly filled with doubts until the moment their child arrives. In other cases, the pregnancy can be associated with bad news: the child isn't viable or is suffering from a serious malformation. During scans, particular attention is paid to the nervous system and especially the brain, which are regularly measured and checked. The highly complex development of the nervous system is well understood overall but there are still many grey areas regarding the mechanisms that govern the workings of this sophisticated machinery. And it is these grey areas that Laurent Nguyen's team is endeavouring to clarify by studying the differentiation and the migration of the neurons in the cortex. In 2009, the researchers from Liège, along with Alain Chariot's team, showed that the Elongator complex played an important role in the migration and differentiation of the neurons in the cortex (see article Neuron migration "under the wing" of Elongator). What they didn't realise at the time was that this discovery would lead them to pin down the mechanism which, unbeknown to them up until now, plays a role in the early stages of the cerebral cortex's development.

The absence of Elongator and microcephaly

"While we were working on Elongator, we used the in utero electroporation technique to reduces the expression, and therefore activity, of Elongator in a limited number of cortical cells", explains Laurent Nguyen, Director of the Laboratory of Molecular Regulation of Neurogenesis, GIGA-Neurosciences. "What's surprising is that Elongator is expressed through the cortical wall starting from the progenitors, along the ventricle, up to the neurons which are in the process of migrating and differentiating. However, with in utero electroporation, we observed that the reduction in Elongator expression modified the migration and differentiation of the neurons without impairing the biology of the cortical progenitors". Although Elongator is expressed in the cortex's progenitors and stem cells, why does reduced Elongator activity only affect the behaviour of the neurons? That's the question the scientists wanted to find an answer to. "We used another strategy: transgenic mice in which the absence of the enzymatic subunit of the Elongator complex abolished the activity of this complex in both the stem cells and neurons in the cortex", Laurent Nguyen points out. The mice in question didn't "only" present a migration and differentiation defect in the cortical neurons, as is the case in rodents that have been subjected to in utero electroporation. The transgenic mice were far more severely affected: they suffered from microcephaly. "This is what we showed in an article published in October 2015 in Developmental Cell, a Cell Press journal", the researcher elaborates (see article The secrets of microcephaly are revealed).

UPR helps overcome cellular stress 

While microcephaly can also be characterised by migration defects in the neurons, it is mainly due to an overall reduction in the number of neurons within the cortex. Either because the progenitors don't produce a sufficient amount of them, or because they have difficulty surviving. "Ultimately, the percentage of neurons is reduced and this leads to microcephaly", Laurent Nguyen continues. "These observations have allowed us to conclude that Elongator plays an important role in both types of cells: the progenitors and the neurons. Elongator is indeed involved in the neurons' ability to migrate and differentiate, and allows adequate production of neurons by the progenitors". When Elongator is no longer functional in the cortical wall cells and in particular in the progenitors, this causes cellular stress. "Molecular analyses clearly show signs of cellular stress that is conveyed through stress in the endoplasmic reticulum", the scientist adds. The endoplasmic reticulum (ER) is one of the organelles responsible for producing and modifying a large number of proteins in the cell. Stress can occur in the endoplasmic reticulum owing to the accumulation of misfolded proteins, for instance. The latter either have to be degraded or correctly refolded with the help of chaperone proteins. "That's where the Unfolded Protein Response (UPR) comes in", Laurent Nguyen explains. "This response will rectify the problem by producing chaperones to properly refold the proteins or by activating autophagy signalling pathways to get rid of the misfolded proteins".   

When the neurons go missing

So, to summarise: when Elongator is inactive, cellular stress occurs and the UPR pathway is activated. "However, excessive activation of UPR impairs neurogenesis", Laurent Nguyen reveals. "Stem cells with excess UPR activation will tend towards direct neurogenesis rather than indirect neurogenesis". This means that in the mouse's cortex, the apical progenitors surrounding the ventricle can give birth to neurons directly or indirectly. At the beginning of corticogenesis, the cells that divide will generate a stem cell and a neuron (direct neurogenesis). But as corticogenesis progresses and the need for neurons increases, these stem cells will choose another method of differentiative division. Rather than producing a neuron, they will tend to produce an intermediate progenitor that is able to proliferate before giving birth to neurons, ultimately amplifying the total number of neurons produced (indirect neurogenesis). "When Elongator doesn't function, there is a reduction in the pools of intermediate progenitors and the neuron amplifier is lost. That's why there are less neurons in the cortex in the end and this translates into microcephaly", Laurent Nguyen explains. 

The molecular mechanism that controls the choice of the stem cell's differentiative division was still unknown up until now. Why and how does this cell chooses to give birth to a neuron and a stem cell or an intermediate progenitor and a stem cell? The literature still provided no answer to this question. 

Forcing the amplification of neurons

"We asked ourselves whether the UPR had a physiological function for the control of this transition during corticogenesis", the scientist continues. "And this is indeed the case! We isolated the apical progenitors at different stages of development and we showed that there is indeed an UPR pathway that is more active at the beginning of corticogenesis. As the cortex continues to develop, this signalling pathway's activity diminishes". Hence, the UPR is very active during direct neurogenesis and is less so during indirect neurogenesis. In order to test the potential role of UPR in this balance between direct and indirect neuron production, the scientists targeted the UPR pathway early on. "We wanted to see whether this would alter the differentiative division capacity of stem cells because at this stage, almost all of them should produce neurons directly", Laurent Nguyen clarifies. "Well, if we reduce the intensity of the UPR pathway at the beginning of corticogenesis, the progenitor is forced to give birth to intermediate progenitors rather than a neuron!". Therefore, there is a physiological UPR pathway that controls the transition from direct neurogenesis behaviour to indirect neurogenesis behaviour. It is precisely the importance of the physiological UPR in controlling corticogenesis that the researchers revealed in an article published in the journal Trends in Neurosciences (1). They also gathered all related information concerning other systems such as the contribution of UPR in the development of drosophila neuroectoderm, the choice of receptors during the formation of the olfactory bulbs, etc. "There are many things in the literature that support the idea that a signalling pathway, which we believed was just a homeostatic response to stress, could actually play a key role in the development of the nervous system", Laurent Nguyen emphasises.  

UPR and foetal stress 

The scientists went one step further. The absence of Elongator creates foetal stress in mice, and foetal stress can be accompanied by microcephaly, as is the case in foetal alcohol syndrome, which affects some babies whose mother drank alcohol during the pregnancy. "These children have a flat midface, small eye openings, a short flat nose and, above all, microcephaly", Laurent Nguyen says. "Alcohol creates cellular stress which could also include stress in the endoplasmic reticulum in vivo. However, no-one has yet shown that the microcephaly in these children could result from a deregulation of the UPR signalling pathway. This is something that we are currently testing", he points out. Another line of research chosen by the scientists to test their model is that of foetal stress induced by viral infection. Starting out with purely fundamental research, the scientists have initiated more translational research. "It clearly shows the importance of funding non-oriented research", the researcher is keen to stress.

With these discoveries, Laurent Nguyen and his team have opened a very broad field of research in order to understand how UPR signalling is involved in the development of the nervous system. "Now it's a question of knowing what regulates this signalling pathway upstream. Is it stress or not? For now, we don't know how to connect the involvement of UPR upstream and downstream in brain development", Laurent Nguyen continues.

Defining the role of this signalling pathway could provide a new target in the long term for treatments against congenital disorders such as microcephaly, as well as various disorders of the nervous system. For instance, the UPR pathway is activated in the majority of neurodegenerative diseases such as Parkinson's, Alzheimer's, and amyotrophic lateral sclerosis… 

(1) Godin J, Creppe C, Laguesse S, Nguyen L.. Emerging Roles for the Unfolded Protein Response in the Developing Nervous System. Trends Neurosci. 2016 Apr 26. pii: S0166-2236(16)30002-9. doi: 10.1016/j.tins.2016.04.002.