Psidin is required for neuron survival and axon targeting through two distinct molecular mechanisms in Drosophila

The formation of neuronal networks depends on the proper
development and targeting of the neurons within the network. One
key challenge during the development of such networks is the
correct cross linking of axons and dendrites. Only correct synapse
formation between dendrites and axons will allow neurons to
contribute to the entire network. Therefore further insights into
axon targeting mechanisms will help to understand the underlying
developmental processes and contribute to future cures for a number
of related diseases. Generally, once a neuron forms an axon, it
starts growing towards a certain “target zone”. The underlying axon
targeting mechanisms are controlled by a large number of
extracellular cues provided by the extracellular matrix and
neighboring cells. Depending on the neuron type, axons travel
different distances towards their future synaptic partner. During
that journey the neurons, more specifically the growth cone,
constantly comes into contact with guidance cues. The growth cone
symbolizes the forefront of an axon and is responsible to integrate
different guidance signals. Depending on their nature, they trigger
the local assembly or disassembly of the cytoskeleton and
ultimately force the axon to turn into a certain direction.
Although different guidance cues activate different signaling
pathways, all of these cascades will eventually converge down on
the cytoskeleton. These cytoskeletal rearrangements and changes in
actin dynamics within the growth cone will promote the turning of
the entire axon. In a series of events different guidance cues,
attractive and repulsive, will guide the growth cone to its
respective target. In this study I used the olfactory system, more
specifically the olfactory receptor neurons (ORNs), of Drosophila
melanogaster to investigate the mechanisms of axon targeting. The
olfactory system of the fruit fly proved to be a very powerful
model organism for a number of reasons: First, the number of
genetic tools available for Drosophila allows the manipulation of
many cellular aspects. Second, ORNs have an extremely stereotyped
targeting pattern that proved to be a good system to investigate
axon targeting mechanisms. The work presented in this thesis
studied the role of the highly conserved actin binding protein
Psidin during the development and targeting of ORNs. Herein, I was
able to demonstrate that Psidin uses two independent molecular
mechanisms to control ORN targeting and survival. To elucidate
Psidin’s role in the aforementioned processes, I analyzed two
predicted null alleles psidin1 (Brennan et al., 2007) and
psidin55D4 (Kim et al., 2011), and one hypomorphic allele
psidinIG978 (this study). The new hypomorphic allele psidinIG978
was mapped during this study and found to have a single point
mutation within Psidin’s coding region (E320K). The data shown in
this study demonstrate that Psidin is required at two different
time points during the development of the olfactory system. During
ORN development, Psidin is required as non-catalytic part of the
N-acetyltransferase complex (NatB) to ensure ORN survival. At later
stages during development, Psidin functions as an actin binding
protein to regulate actin dynamics to ultimately ensure proper ORN
axon targeting. I was able to show for the first time that Psidin’s
previously reported function as actin binding protein in oocytes
(Kim et al., 2011), is also true for neurons. The loss of Psidin
leads to significantly reduced lamellipodia in growth cones of
primary neurons in vitro. In agreement with Psidin’s role in actin
dynamics is the finding that the parallel removal of the actin
stabilizer Tropomyosin rescues the lamellipodia defect in psidin1
primary neurons. This strongly argues for Psidin being an actin
destabilizing protein and antagonist of Tropomyosin. In general,
psidin1 and psidin55D4 mutant axons showed severe mistargeting
defects in vivo – e.g. defasciculation in Or59c and Or42a neurons
or ectopic synapse formation in Or47a neurons. However, axons
mutant for psidinIG978 displayed a less severe phenotype compared
to the null alleles. In agreement with in vitro data, the parallel
removal of Tropomyosin rescued the targeting defect in Or59c
neurons in vivo. The growth cone and the lamellipodia are both
important structures that keep axons responsive towards guidance
cues. Therefore the lamellipodia reduction in psidin mutants is
likely the cause for the observed targeting defects. Nevertheless,
Psidin is required differentially among the ORN classes – the ones
that project to dorsolateral or ventromedial glomeruli within the
antennal lobe (AL) are more affected than centrally projecting
classes. ORN classes that are more affected in psidin mutants have
to turn upon entry of the AL. Therefore those classes (dorsolateral
and ventromedial) have a higher requirement of Psidin, which has to
maintain the lamellipodium, so that the axon can respond to cues in
the first place. In addition, I overexpressed different isoforms of
LimK and Cofilin to artificially create conditions that favor actin
stabilization or destabilization. More generally, conditions that
promoted actin destabilization and actin stabilization were able to
rescue and aggravate the psidin1 phenotype, respectively. In
addition to the targeting defect, psidin1 and psidin55D4 mutants
showed a strong reduction in ORN cell numbers. In contrast, cell
numbers were not affected in psidinIG978 mutant flies. Again, ORN
classes were affected differently – e.g. Or42a neuron number was
reduced by 83%, but Or59c number was only reduced by 46%.
Indicating Psidin’s function in ORN survival, the expression of the
anti-apoptotic protein p35 in psidin mutant neurons selectively
rescued the cell number, but failed to rescue the targeting
defects. Interestingly, the Psidin/Tropomyosin double mutant showed
the opposite effect; here the targeting was rescued, but not the
cell number. These findings gave strong indications that Psidin has
two independent functions during ORN targeting and development.
Psidin is predicted to be the non-catalytic part of the
N-acteyltransferase complex B (NatB) in Drosophila (Brennan et al.,
2007). Here, Psidin (non-catalytic) forms the NatB-complex together
with dNAA20 (catalytic). This complex is thought to acetylate
nascent protein chains N-terminally. In this study I demonstrated
for the first time that both proteins interact in vivo and in
vitro. Indicating that the NatB-complex is involved in ORN
survival, the knock-down of dNAA20 in psidinIG978 mutants led to a
reduction of ORN cell number that is reminiscent of the cell number
in psidin1 or psidin55D4 background. At the same time, the
knock-down of dNAA20 had no effect on the targeting of ORNs.
Furthermore I was able to show that wild type Psidin and
PsidinIG978 interact with dNAA20 at comparable levels in vitro.
This is in agreement with the finding that the psidinIG978 allele
selectively affects ORN targeting, but not ORN survival. In
addition, I was able to map the interaction domain between Psidin
and dNAA20. This revealed that the point mutation found in
psidinIG978 is just outside of the minimal interaction domain.
Deletion of the entire interaction domain led to a complete
abolishment of the Psidin/dNAA20 interaction. Furthermore I was
able to demonstrate that the interaction of Psidin and dNAA20 is
regulated by the phosphorylation of a highly conserved serine
residue (S678). Expression of the non-phosphorylatable Psidin
isoform (S678A) rescued the targeting and cell number phenotype in
vivo. Contrary expression of the phosphomimetic isoform (S678D)
only rescued the targeting phenotype, but failed to restore ORN
cell number in vivo. In line with this observation is the finding
that the S678D isoform is unable to bind dNAA20 in vitro. At the
same time the S678A isoform binds dNAA20 at normal levels in vitro.
Taken together, the data presented in this work demonstrate that
Psidin has two functions during the development and targeting of
ORNs using two independent molecular mechanisms: First, during axon
targeting Psidin is required as an actin destabilizing molecule and
antagonist of Tropomyosin. Psidin maintains the lamellipodia size
in growth cones and keeps the cytoskeleton in a dynamic and
responsive state. This ensures that growing axons can respond
properly to various guidance cues. Second, to ensure ORN survival,
Psidin is required as non-catalytic part of the NatB-complex. Here,
Psidin interacts with the catalytic subunit dNAA20. The formation
of the NatB-complex is regulated by phosphorylation of a conserved
serine. In its unphosphorylated state Psidin binds dNAA20 and
ensures ORN survival, whereas phosphorylation causes the
abolishment of this interaction which results in a reduction of ORN
cell number. Concluding, this thesis unambiguously shows that
Psidin is required at different time points during the formation of
the olfactory system of Drosophila. It utilizes two different
pathways to ensure (i) ORN survival as part of the NatB-complex and
(ii) ORN targeting as actin binding protein. Due to its strong
conservation in higher organisms, the here presented data provide
important insights into the function of Psidin’s mammalian
homologues.