Neuronal development is a very finely controlled process. Different proteins act together for the formation of correct neuronal networks, among these: Synapsin III (SynIII). SynIII is involved in axonal elongation and the formation of a growth cone, the structure at the tip of a growing axon responsible for guiding axon outgrowth. SynIII is early expressed during development, and mutations of this protein have been associated with disorders linked to neuronal development, such as schizophrenia. Migration defects are often seen in neurodevelopmental disorders, and low levels of SynIII and an altered pathway of SynIII are found altered in patients with schizophrenia.
So many clues led to hypothesize a role of Synapsin in neuronal development.
During brain formation, neurons are usually born in different areas of the brain and only afterwards, they migrate towards their final area and form highly organized structures that are crucial for a correct brain functioning. The cortex is a region of the brain organized in several layers, each layer is generated at different time points during development.
SynIII Knock Down. A way to check for the role of a protein in a specific cell process is to remove this protein and analyze the consequences on cell physiology.
A very common way to do it is by using short hairpin RNAs (shRNAs). These are small sequences of RNA that are complementary to the mRNA coding for a certain protein (mRNA is the molecule keeping the information to “build” a new protein). Due to their complementarities, once these small RNAs will be expressed in the cells, they will bind to the mRNA, making it double strand. Since mRNA is usually a single strand, when the cell will recognize that there is double strand mRNA (due to the anneal with the shRNA), a specific cell signaling will be started in order to eliminate it: the cell will degradate this part of the mRNA and no protein will be synthesized. So the overall expression of the target protein will be largely decreased. This method is called Knock Down (KD).
These shRNAs can be introduced in the very early stages of a mouse development, by a technique that is called in utero electroporation. Basically, the shRNAs are injected in the uterus, inside the embryos, in the area where there are progenitor cells of pyramidal neurons (these progenitors cells will give birth to pyramidal neurons, which are neurons of the cortex) and then led into the cells by applying an electric field. The molecule will travel, driven by the electric field, into the cells. Then they analyzed the migration of these neurons that, as they express the shRNA, have very low levels of SynIII.
Even if there was no effect on neuronal proliferation or differentiation, neuronal migration was significantly delayed when SynIII levels were lower. Some cells were misplaced in the “wrong” layers of the cortex, and were “misoriented”. A neuron is a very polarized cell, meaning that each “side” has a defined role for neuronal physiology and has to be specifically oriented. The correct orientation is very finely controlled during development. The absence of SynIII affected in a certain extent also the correct orientation of these neurons.
Rescue. In order to check if the effects observed with shRNAs were really due to downregulation of SynIII, they made what is called a rescue: so the protein was re-introduced. The cells were injected with both the shRNAs against SynIII and the DNA sequence coding for SynIII, resistant to the shRNA itself (see appendix for details): the cells were “rescued” and did not show any delay in migration and no defects in cell orientation.
SynIII Knock Out. SynIII knock out (KO) mice, meaning transgenic mice lacking the gene for SynIII (so having no protein at all since the beginning of the development, while with shRNAs protein expression is just acutely downregulated at a certain stage during development) show some behavioral defects. An accurate analysis of the phenotype, displayed migration defects of pyramidal neurons and a slight misorientation (less strong than in KD).
SynIII overexpression. Increasing the level of a protein disrupts the delicate equilibrium required for a correct cell physiology. So what happens if we overexpress SynIII? Some cells were actually misplaced in the wrong layers of the cortex, like it happened in shRNA-treated animals (even if there was no effect on cell orientation).
What about the mechanism?
Semaphorin3A (Sema3A) is a guidance molecule involved in neuronal migration during development of the cortex. When Sema3A binds to its receptor Neuropilin1 (NP1), the receptor associates with its co-receptor PlexinA2 and activates Fyn kinase. Kinases are proteins that modulate the activity of their targets by adding phosphate groups. After activation, Fyn recruits CDK5 to plexinA2. Activated CDK5 in turn phosphorylates SynIII, and indeed, the level of phosphorylation of SynIII was increased after stimulating the cells with Sema3A.
How does this result into altered migration and neuron orientation?
Synapsin are known to bind vesicles, regulating their trafficking, and to cytoskeleton. Cytoskeleton is an internal “skeleton” of a cell that is made of two main elements: actin and tubulin proteins assembled in a very organized fashion. Cytoskeletal dynamics and vesicle trafficking are involved in membrane rearrangements that cells undergo during cell migration. Altered SynIII activity could influence cytoskeleton dynamics, ultimately affecting neuron migration.
Conclusion.
Cell physiology is the result of a very delicate and intricate “net” of different processes, and is fascinating to think how interfering with it at the single cell level has visible effects on the whole organism.
Altering the normal protein levels of SynIII, with either overexpression, knock down or Knock out, affects neuronal migration, due to an altered response of the cells to the guidance molecule Sema3A, and it might be related to the symptoms of schizophrenia.
In vivo experimentation has often received a lot of critics. However, as we have seen in this work, while single cell physiology can be investigated in vitro, the effect on the building of neuronal network –as a consequence of cell physiology-, which is crucial in understanding the pathological basis of several disorders, needs to be explored in vivo. Only understanding the key role of proteins involved in neuronal development will allow to identify good targets to treat some disorders linked to development.
(The paper of Perlini et al. has a very nice figure -Figure 7- summarizing the mechanism described in this article)
APPENDIX
RNA-mRNA and shRNA. RNA is a nucleic acid, a molecule required to decipher the genetic information contained in the DNA. mRNA is a type of RNA that has the information to guide the synthesis of a new protein. shRNA are short sequences of RNAs used to manipulate protein expression.
shRNA-resistant DNA. How can we reintroduce the DNA coding for a protein, without risking to knock it down again with the shRNA that we use to decrease the expression of the endogenous protein? To have shRNA working, it has to be perfectly complementary to the target mRNA. So if the DNA for SynIII that we re-introduce for the rescue has few mutations, the mRNA coming from this DNA (we will call it exogenous mRNA) will have few mutations that will prevent the binding of shRNA. So the exogenous mRNA will not be degradated and the protein will be synthesized.
The few mutations will not affect the protein coming from the re-introduced DNA? No, because we can introduce mutations that do not result into a change of the protein. How? This can be done thanks to the “redundancy of the genetic code”. What does it mean? Proteins are made of aminoacids (that we can call “the bricks of the proteins”). The aminoacids combine in different ways to make all the different existing proteins. Every aminoacid is coded by a specific combination of 3 nucleotides that compose the mRNA, a codon. The combination of nucleotides determines the type of aminoacid. Eg Arginine is coded by the codon AGA= adenine, guanine, adenine. However, the codon AGG also codes for an Arginine. So if the shRNA will be complementary to the AGA sequence, if we mutate AGA into AGG, this mutation will not affect the aminoacid -and so the protein synthesized-, but it will prevent the binding of the shRNA to the mRNA, which will no more be complementary.
Reference: Synapsin III acts downstream of semaphorin 3A/CDK5 signaling to regulate radial migration and orientation of pyramidal neurons in vivo. Perlini LE, Szczurkowska J, Ballif BA, Piccini A, Sacchetti S, Giovedì S, Benfenati F, Cancedda L. Cell reports 2015