The role of PTEN in the formation of thalamocortical axonal tracts
Heleen van ‘t Spijker1
1
Utrecht University, graduate school of life sciences
Abstract
Autism spectrum disorder (ASD) is a heritable disorder which affect ̴1% of the population.
Unfortunately, no targeted therapy is available. It is widely established that PTEN is important for
neuronal development and its molecular pathways are a promising potential target for the
development of treatments for ASD. Since disruption of the connectivity between the thalamus and
the cortex is known to have a role in ASD, new insights in the regulation of the formation of axonal
tracts from the thalamus to the cortex by PTEN are required. An overview of the current knowledge
of the influence of PTEN on axon development is here provided. Furthermore, the involved molecular
pathways are discussed. Based on current knowledge, it is hypothesized that PTEN decelerates
axonal outgrowth and enables binding of the axon to its target. Starting points to investigate the
details of the function of PTEN in the development of the thalamocortical tract are described. In the
end, this review provides insights which make the discovery of new targeted therapies for ASD
possible.
Introduction
Autism spectrum disorder (ASD) is a group of severe neurodevelopmental disorders for which no
sufficient therapies are available1,2. The main symptoms are communication deficiencies and rigidity
in social behavior. Patients also suffer from many comorbidities, including seizures, anxiety disorders
and depression. These symptoms are presumably caused by miswiring of the brain; the brain fails to
prioritize amongst different forms of sensory information. Since ASD is associated with a problem of
connectivity3, 4, it is no surprise several risk genes for ASD, such as phosphatase and tensin homolog
deleted on chromosome 10 (PTEN), have a role in the formation of axonal tracts. In this review, an
overview is provided of the current knowledge of the role of PTEN in the morphology of the brain
and the molecular pathway of PTEN in axon tract formation. The focus is put on the formation on the
thalamocortical axons, because the cortex is responsible for the higher brain functions which are
disrupted in ASD and the thalamus organizes and prioritizes the information input for the cortex.
Despite the thorough research that has already taken place to investigate the role of PTEN, its role in
the development of this tract remains unknown. Based on the overview provided in this review a
new hypothesis on the role of PTEN in the formation of the thalamocortical tract is formulated.
Criteria to validate this hypothesis are provided. Investigations on the basis of these criteria may lead
to the development of new targeted treatments for ASD. In this review I analyze the current
knowledge in PTEN research with a particular focus on the formation of the thalamocortical tract, in
order to provide new starting points for directed ASD research.
1
Currently, the molecular pathway of PTEN and the function of PTEN in the development of
the brain are roughly known. PTEN is an enzyme which catalyzes the dephosphorylation of
phosphatidylinositol-3,4,5-triphosphate (PIP3) into phosphatidylinositol-4,5-biphosphate (PIP2)5. It
antagonizes the phosphorylation step performed by phosphatidylinositol 3 kinase (PI3K), which
causes phosphorylation of PIP2. PIP3 phosphorylates and thereby activates Akt (figure 1). The
downstream PI3K/AKT pathway is important for several cellular processes, such as proliferation and
migration of cells. PTEN was originally discovered as a tumor suppressor protein6. Loss of PTEN may
cause cancer, but it may also result in several neurological conditions such as macrocephaly, seizures,
mental retardation and ASD. Germ line deletion of PTEN in mice leads to death at the embryonic
stage7. For this reason, to investigate the function of PTEN, several conditional PTEN knockout mouse
models have been developed. The conditional deletion of a gene allows researchers to investigate
the effect of PTEN in a specific tissue or cell type. Conditional deletion means the expression of a
protein, such as PTEN, is coupled to the expression of another protein, specific for the target tissue
or cell type, through the insertion of Cre and Lox sequences in the DNA8. Overall, the conditional
knockout (cKO) models of PTEN display hypertrophy of cells and disrupted lamination of neurons.
However, though the pathway of PTEN is roughly known, the precise role of PTEN in the outgrowth
of axons and the development of synapses remains undiscovered.
With this review I aim to answer how PTEN influences the formation of the thalamocortical
tract. The development of the thalamocortical tract is a complex and highly regulated process, of
which the basis will be delineated in the first section of this review, to provide a fundamental
background for the description of the role of PTEN in this process. In order to provide a starting point
for new targeted experiments to investigate the role of PTEN in the formation of thalamocortical
axon tracts, I provide an overview of the morphological findings in PTEN research and then I describe
the molecular mechanisms in the PTEN pathway. Subsequently, I formulate a new hypothesis about
the role of PTEN in the growth of thalamocortical axons. Then, I explain what the criteria are to
validate this hypothesis. Together, the analyses in this review offer a synopsis of the current
knowledge on PTEN and insight in how new research may lead to the development of targeted new
treatments for ASD.
Figure 1. PTEN pathway. Phosphatase and tensin homolog deleted on chromosome 10 = PTEN,
phosphatidylinositol-4,5-bisphosphate 3-kinase = PI3k, phosphatidylinositol-3,4,5-triphosphate =
PIP3, phosphatidylinositol-4,5-biphosphate = PIP2, pyruvate dehydrogenase kinase 1 = PDK1,
glycogen synthase kinase 3 beta = GSK3β. Following a signal from outside the cell, PI3K
phosphorylates PIP2 into PIP3, which then activates PDK which in turn activates Akt. PTEN catalyzes
the reverse step; from PIP3 to PIP2, thereby deactivating Akt.
2
Development of the thalamocortical tract
In this review, the focus is put on the development of the thalamocortical tract. This focus is chosen
because the communication between the thalamus and the cortex is thought to have a key role in
autism spectrum disorders9,10. The key function of the thalamus is the filtering of sensory input for
the cortex, prioritizing which input is most relevant. One of the prominent theories on ASD suggests,
that in patients it is the failure of exactly this prioritization that causes the disorder9. Indeed,
underconnectivity between the thalamus and the cortex was found in ASD patients9. Furthermore,
the thalamocortical tract provides a great opportunity to investigate axonal tract formation. The high
amount of previous experiments with the formation of this tract provides good comparison material
for new findings and the formation process is very complex and highly regulated. For these reasons,
the formation of the axonal tracts from the thalamus to the cortex is a promising guide to gaining
new insights in the ASD brain.
E13.5
E18.5
Figure 2. The development of axon tracts between the cortex and the thalamus. AT E13.5 axons
from the thalamus start growing to the cortex and simultaneously axons from the cortex start
growing towards the thalamus. The thalamic axons leave the thalamus and grow though the
prethalamus. The cortical axons grow to the pallial-subpallial boundary and pause there. At E18.5
thalamic axons are located at the subplate of the cortex and the cortical axons have reached the
thalamus10.
In order to investigate the function of PTEN in the development of the thalamocortical tract an
overview of this process in the healthy mouse brain first needs to be provided. The neurons in the
thalamus extend their axons to the cortex via a strict route. Firstly, thalamocortical projections
descend trough the ventral thalamus and pass through the internal capsule. The projections then
cross the striatocortical junction. Subsequently, the projections reach their target in the cortex
(figure 2)11. The passing of the prethalamus and the internal capsule takes place at E13. Interestingly,
the development of axonal tracts takes place synchronously with the growth of the corticothalamic
projections. The simultaneous growth allows for the first meeting of the thalamocortical axons and
the corticofugal projections to take place when they have passed the internal capsule, at E14.5. At
E15.5 the thalamic axons pause at the subplate11. Invasion of the target layer and synapse formation
occur postnatally12. The formation of the thalamocortical axon tract is a strictly ordered process.
3
To analyze the function of PTEN in the formation of the thalamocortical tract a description of the
regulation of this process is necessary. Axons from the thalamus are regulated by several methods.
Guidepost cells with axons extended along the route provide guidance for the thalamocortical
axons13. These pioneer neurons are localized in the reticular nucleus of the prethalamus, which will
later become the internal capsule. Furthermore, the organization of the thalamus itself has a key role
in the regulation of the growth of the thalamic axons. The thalamus is divided in several nuclei, such
as the posterior nucleus (Po), the dorsomedial nucleus, the pulvinar nucleus and the ventral
posteromedial nucleus (VPM). Axons from a distinct nucleus stay together and invade the same
location of the cortex12. During their waiting period at the subplate the thalamic axons also form a
topographic map11. Moreover, the thalamocortical axons regulate the organization of the cortex. The
input from the VPM and the Po in the thalamus leads to barrel formation in the cortex14. These
barrels are columns of the cortex and each represents sensitive input from a spatially distinct area of
the body. In conclusion, the development and the organization of the thalamocortical tract are highly
ordered, and the thalamocortical tract is an ideal model to investigate PTEN activity.
Morphology
Research with mouse models in which PTEN is (conditionally) deleted has led to the identification of
morphological aberrations caused by PTEN loss. The creation of conditional PTEN knockout models,
of which an overview is provided in table 1, was necessary because germ line deletion of PTEN results
in death before birth7. Investigation of the embryos of total knockout mice led to the conclusion that
cellular patterning was disturbed both caudal and cephalic, where the cells which will later form the
brain are localized. Since the embryos die before they reach a stage where different brain regions
can be distinguished, no conclusions can be drawn about brain development based on these findings.
However, mice heterozygous for PTEN are viable. Heterozygous expression of PTEN leads to
disrupted social discrimination and circadian rhythm15, which proves that PTEN is crucial for normal
brain function. Moreover, both PTEN heterozygous mice and many of the cKO mice show
macrocephaly15, 16. Expression of PTEN is crucial for the development of the brain.
The changes in the functioning of the brain and the size of the brain caused by PTEN deletion
raise the question how individual brain cells are influenced by PTEN deletion. Cell growth and
reproduction of several cell types in the brain are decreased by PTEN. Neural progenitor cells are the
cells from which all neurons develop. When PTEN depletion is caused through Nestin, which is
expressed specifically in neural progenitor cells, loss of PTEN causes cells to go through more cell
cycles17. Moreover, loss of PTEN enhanced self-renewal capacity. When PTEN was deleted postnatally
in Nestin expressing cells, the amount of cells in the neural tube increased18. Another important cell
type in the brain is the glia cell, which provides support and maintains homeostasis in the brain.
Several research groups determined loss of PTEN in glial fibrillary acidic protein (GFAP) expressing
cells leads to increased soma size16, 19. Additionally, both nuclei and mitochondria of GFAP-positive
cells are enlarged when PTEN is absent20. Similar results were found when PTEN was deleted
temporarily in dentate granule cells. After PTEN deletion the soma size of the dentate granule cells
increased21. Furthermore, when PTEN was deleted in oligodendrocytes, by using the specific
expression of oligodendrocyte lineage transcription factor 2 (Olig2), it was again established PTEN
slows cell reproduction22. It was found that loss of PTEN in these cells causes more cells to enter the
cell cycle and they no longer remain in the G0 stage. These results lead to the conclusion that PTEN
functions as a brake on cell proliferation; loss of PTEN causes cells growth and replication to increase.
4
Role of PTEN
investigated
Brain as a
whole
Type of PTEN
depletion
PTEN -/PTEN +/-
GFAP
Nestin
Postnatally
Nestin
GFAP
GFAP
Cell
GFAP
development shRNA in
hippocampus
Olig2
En2 / L7
Nex
Cell
migration
and brain
structure
development
Retinal
progenitors
L7
GFAP
GFAP
DAT
Nestin
Nex
RNA
interference
shRNA in
Axon and
hippocampus
dendrite
Postnatally
development
Camk2a
RNA
interference
RGC
Morpholino
auditory cortex
Synapse
development
GFAP
GFAP
Nse
shRNA in
hippocampus
Main results
Author
Embryonic lethality
Social discrimination and circadian rhythm
impairment
Death at week 29 and macrocephaly
Increased proliferation of neural stem cells
Expansion of the sub ventricular zone and
increased cell number in the glial tube
Increased soma size
Increased soma size
Nuclei, soma and mitochondria enlarged
Increased cell size
Suzuki7
ClippertonAllen15
Backman16
Groszer17
Zhu18
Aberrant neurogenesis and oligodendrocyte
loss
Reduced proliferation in En2 model , not in L7
model
Less compact and distorted layers, and large
neurons in marginal zones
Decreased layering of retinal ganglion cells and
loss of adhesion junctions
Purkinje cells show bad migration and
positioning
Neuronal migration defect
Lamination disrupted in cerebellum,
hippocampus and cerebral cortex
Fiber density increase
Neural sphere increase
Thicker neurites
Dendritic arborization 25% increase
Maire22
Increased spinal density and sEPSC frequency
increase
New dendrite development possible in adults
in layer 2-3 of cerebellar cortex
Axonal outgrowth and growth cone size
increase in motor neurons
Long-distance axon regeneration is possible
Chemorepulsion but not chemoattraction is
dependent on PTEN
Hyperconnectivity of local and long range
connections
Ectopic dendrites and axons
Increased synaptic vesicles and presynaptic
terminals enlarged
Synaptic plasticity dysregulation, before
morphological defect
Increased spinal density
Luikart21
Backman16
Kwon19
Fraser20
Luikart21
Marino23
Kazdoba24
Sakagami25
Marino23
Yue26
Wen27
Diaz-Ruiz28
Groszer29
Kazdoba24
Jaworski30
Chow31
Ning32
Park33
Henle34
Xiong35
Kwon36
Fraser20
Takeuchi37
Luikart21
5
Table 1. Conditional PTEN knockout experimental findings. Phosphatase and tensin homolog
deleted on chromosome 10 = PTEN, glial fibrillary acidic protein = GFAP, oligodendrocyte lineage
transcription factor 2 = Olig2, engrained homeobox = En2, purkinje cell protein 2 = L7, neuronal basic
helix-loop-helix protein = Nex, dopamine transporter = DAT, calcium/calmodulin-dependent protein
kinase 2 alpha = Camk2a, neuron specific enolase = Nse, short hairpin RNA = shRNA retinal ganglion
cell = RGC.
However, contradicting results about the effect of PTEN on proliferation have also been found. PTEN
deletion in engrained homeobox (En2) expressing cells, which are located at the border of the
hindbrain and the midbrain, leads to a decrease in proliferation23. The researchers who performed
the experiment with the En2 depleted animals provided a potential explanation for this discrepancy:
in their experiment the development of the cerebellum was disturbed, which could lead to a change
in the amount of growth factors the cerebellum excretes towards the En2 positive cells. When they
investigated PTEN deletion in purkinje cell protein 2 (L7) expressing cells, a protein specific for
Purkinje cells, they replicated the finding that PTEN down regulates proliferation23. Overall, these
results indicate PTEN is indeed important for proliferation of cells.
The alterations found in individual cells under the influence of PTEN lead to the question how
PTEN depletion influences how the cells migrate. When PTEN is deleted conditionally through the
neuronal basic helix-loop-helix protein (NEX), layers in the neocortex are distorted and less
compact24. Moreover, large neurons migrate into the marginal zone, where they are not located in
healthy control animals. This finding indicates cortical neuronal migration depends on PTEN. Similar
results were found when PTEN was deleted in retinal progenitors. Loss of PTEN disturbed layering of
retinal ganglion cells25. Interestingly, in this model a loss of adhesion junctions was identified, which
may provide insight into the mechanism behind the disturbed migration, since cells need to
recognize and bind each other for proper localization. Likewise, in the above mentioned conditional
PTEN knockout model in En2 expressing cells, the migration of Purkinje cells was disrupted23. Several
research groups have studied the influence of PTEN deletion in glia cells, which regulate migration of
neurons. Yue et al. (2005) established that a neuronal migration defect occurs in the cerebellum
when PTEN is deleted in glia cells through the GFAP promoter26. Wen et al. (2013) later identified a
laminarization defect in the hippocampus and the cerebral cortex as well as the cerebellum27. Glia
cells are known to provide a scaffold for neurons for the directions of their migration and these
results show that they require PTEN to provide this guidance. PTEN has a crucial role in the migration
of cells during the development of the brain.
Besides the migration defects of whole cells, zooming in on particular parts of the cells,
investigations on the role of PTEN in the development of axons and dendrites have also been
performed. Several conditional knockout models of PTEN show an increase in neurite development.
For example, when PTEN was deleted in the dopamine neuron system with the dopamine
transporter (DAT) transgenic mouse line, fiber density was increased in the caudal striatum28.
Similarly, in the above mentioned cKO model of PTEN in Nestin expressing cells, an increase in
neuronal spheres was reported29. In a cKO model in which PTEN was deleted in the neocortex,
neurites were also found to be thicker24. Comparable results were found when PTEN was deleted
with other methods. When PTEN was deleted in cultured neurons by the use of RNAi, a 25% increase
in total amount of dendrite tips was found and the dendrites were more elaborate30. Together, these
results indicate absence of PTEN leads to an increase in neurite development.
6
More detailed investigations of the role of PTEN in the development of neurites have been
performed. The influence of PTEN on spinal outgrowth is not limited to the developmental period.
When PTEN was conditionally deleted in the calcium/calmodulin-dependent protein kinase 2 alpha
(CAMK2A) promoter postnatally, new dendrite development was identified in layer 2-3 of the
cerebral cortex31. Zooming in specifically on axons, in cultured motor neurons axon elongation and
growth cone size increase when PTEN is deleted32. Furthermore, in axon regeneration research, PTEN
deletion is part of a mix of stimulants and inhibitors that make axon regeneration possible38,39. PTEN
deletion is combined with stimulation by cyclic adenosine monophosphate (cAMP) and oncomodulin
(OCM) to overcome inhibition of axonal regrowth. When this combination was tested on a model of
optic nerve injury, it led to effective full-length axon regeneration and new synapses were formed by
the new axons39. Interestingly, when Henle et al. investigated the role of PTEN in axon guidance, they
found that PTEN is crucial for chemorepulsion but not for chemoattraction34. This finding indicates
PTEN has a specific function in the inhibition of growth of the axon. Moreover, when PTEN is deleted
in axons growing towards muscles, the axonal advance after contact with a muscle is faster40. This
result also indicates PTEN is necessary to inhibit axonal growth, since the axons need to stop growing
in order to form a synapse in the correct location. Together, these results indicate PTEN inhibits
excess axon elongation.
The influence of PTEN on the development of dendrites and axons, leads to the question how
synapse development and synaptic plasticity depend on PTEN. In a cKO model of PTEN in the
auditory cortex, hyperconnectivity was observed. Both local and long range connections were altered
by PTEN deletion35. In a cKO model of PTEN in GFAP expressing cells, it was established PTEN deletion
leads to an increase in synaptic connections20, 36. Furthermore, in this model presynaptic terminals
are enlarged and more synaptic vesicles are present at the synapse20. Interesting results were found
when PTEN was deleted in the neuron specific enolase (NSE) expressing cells of the dentate gyrus.
Synaptic plasticity dysregulation in this model occurs before morphological defects were detectable,
such as hypertrophy in the hippocampus37. This finding indicates synaptic disruptions in cKO PTEN
models may occur even when no morphological disruptions are identified. Moreover, an increase in
spinal density can be caused by PTEN deletion21. Loss of PTEN increases synaptogenesis and disrupts
synaptic plasticity.
Molecular pathway
Besides the investigations of morphological changes caused by PTEN deletion, it is necessary to
determine the molecular pathway by which these effects are caused to clarify how PTEN influences
axonal tract formation. The molecular pathway is crucial to identify targets for therapeutics for
disorders such as ASD based on the function of PTEN. The importance of PTEN for the molecular
mechanisms involved in axonal outgrowth is apparent from the morphological findings described in
the section above. In this section, the molecular pathway of PTEN in axonal tract formation will be
explained. Firstly, I provide an overview of the molecular pathways involved in axon development.
Subsequently, I describe in detail the relationship between the different proteins of the PTEN
pathway and distinct aspects of axon development, such as actin and tubulin organization and an
increase in protein translation. At the end I explain different molecular mechanisms by which PTEN
activity is regulated. Together, these explanations form an analysis of the molecular mechanisms by
which PTEN regulates axon development.
7
Figure 3 PTEN regulates axon elongation pathway. Phosphatase and tensin homolog deleted on
chromosome 10 = PTEN, phosphatidylinositol-4,5-bisphosphate 3-kinase = PI3k, phosphatidylinositol3,4,5-triphosphate = PIP3, phosphatidylinositol-4,5-biphosphate = PIP2, pyruvate dehydrogenase
kinase 1 = PDK1, glycogen synthase kinase 3 beta = GSK3β, drebrin = DBN, Ras-related C3 botulinum
toxin substrate 1 = RAC1, ADP-ribosylation factor 6 = Arf6, collapsing response mediator protein 2 =
CRMP2, tuberous sclerosis protein = TSC, mammalian target of rapamycin = mTor, P70S6 kinase =
P70S6K. Regulation of actin filaments and microtubule, and an increase in protein translation are
necessary to facilitate axon elongation. In response to growth factors from outside the neuron, PI3K
activity is increased. PI3K activity leads to increased translation, and regulation of actin and tubulin.
PTEN catalyzes the reverse reaction of PI3K, which means it decreases the amount of PIP3.
Therefore, PTEN activity inhibits the processes stimulated by PI3K. The downstream effects of PTEN
on its molecular pathway inhibit axon elongation.
8
In order to gain insight in the molecular mechanisms by which PTEN influences axon development it
is necessary to describe the processes which facilitate axon development. For axon development,
growth factors first trigger molecular cascades to elongate axons. When the axon reaches the target,
the molecular balance is changed towards stopping elongation and stimulating the formation of a
synapse. For axon elongation three aspects in the axon have to be regulated; the formation of actin
filaments, the formation of microtubuli and up-regulation of translation. Formation of both actin
filaments and microtubuli enables the cytoskeleton to grow and thus elongate the axon. In order to
facilitate the growth of the cytoskeleton new proteins need to be produced in the axon, which is why
translation needs to be up-regulated. The three aspects are all regulated by the molecular pathways
in which PTEN participates (figure 3)41. In the molecular pathways which regulate axon elongation,
PTEN has the potential to down-regulate the activity. When PTEN is active, the molecular balance
shifts towards the termination of axon elongation. The proteins of the PTEN pathway may be
excellent starting points for the development of new targeted therapies for disorders in which the
PTEN pathway is disrupted, such as ASD. An overview of the available knowledge on the molecular
pathway of PTEN and the role of its proteins in the regulation of axon tract formation will now be
provided.
PTEN inhibits the organization of tubulin into new microtubuli for the elongation of the axon.
PTEN deactivates PDK1 by decreasing the amount of available PIP3. Subsequently, PDK1 cannot
activate Akt, which in turn cannot deactivate GSK342. Indeed, when PTEN is stimulated, the amount
of active Akt decreases and the amount of active GSK3 increases43. The alteration in the amounts of
these proteins coincides with collapse of the growth cone43. This finding indicates PTEN activity is
important for the inhibition of elongation of the axon, since the collapse of the growth cone leads to
a halt in axonal outgrowth. Interestingly, inhibition of one of the downstream proteins of PTEN,
GSK3β, induces the formation of multiple axons. Moreover, in the presence of GSK3β inhibitors, axon
growth and axon branching are increased44. One of the substrates of GSK3β is collapsing response
mediator protein 2 (CRMP2), which in its dephosphorylated form, promotes microtubuli assembly by
binding tubulin45. Deactivated CRMP2 cannot bind tubulin to promote microtubuli polymerization
and stabilization43. In conclusion, PTEN inhibits assembly of microtubuli for axon elongation through
the deactivation of PDK1 which leads to deactivation of CRMP2.
Furthermore, PTEN inhibits the organization of actin in new actin filaments for the elongation
of the axon. The decrease in PIP3 caused by PTEN activity leads to deactivation of Ras-related C3
botulinum toxin substrate 1 (Rac1). Active Rac1 collaborates with ADP-ribosylation factor 6 (Arf6) to
activate Rho, which regulates actin41. The regulation of actin is necessary for the expansion of actin
filaments, which enables the axon to grow. Indeed, the activity of Rac1 and Arf6 stimulates neurite
growth through actin organization46. Interestingly, when PTEN was deleted in motor neurons, an
increase in the amount of actin was found32. This result confirms that one of the functions of PTEN is
the inhibition of actin filament formation. Moreover, two proteins which counteract the activity of
PTEN, Rac1 and PI3K, can form a positive feedback loop in which they activate each other. This
feedback loop is considered a necessary step in axon formation47. The active form of PTEN also
inhibits actin filament expansion through the deactivation of drebrin (DBN). PTEN dephosphorylates
DBN, which causes DBN to become deactivated48. DBN is an actin binding protein which facilitates
the formation of new actin filaments. Together, these findings indicate PTEN inhibits actin filament
formation and thereby regulates the axon elongation pathway.
9
The third aspect of axon elongation which is inhibited by PTEN, is protein translation. PTEN activity
leads to deactivation of Akt. Subsequently, Akt cannot deactivate tuberous sclerosis protein (TSC),
which than cannot deactivate mammalian target of rapamycin (mTOR). Thus, PTEN activity leads to
deactivation of mTor, which causes deactivation of P70S6 kinase (P70S6K). Indeed, when PI3K, the
enzyme for the reverse reaction of PTEN, is inhibited, levels of active P70S6K decrease49. Moreover,
when PTEN is deleted, mTor activity is increased33. Interestingly, when Park et al. compared a PTEN
deletion model with a TSC1 deletion model, they found an increase in P70S6K activity in both
models33. Furthermore, the models showed resemblance in axonal regeneration after optic nerve
crush33. These findings indicate the mTor pathway is important for the effect of PTEN on axons.
Importantly, an increase in actin was found in a PTEN cKO model32, which is an indication of a local
increase in protein translation. In conclusion, PTEN regulates axon growth through deactivation of
the mTor pathway, which leads to a decrease in protein translation.
Discussion of the effects of PTEN on the three aspects of axon development leads to the
question how PTEN activity is regulated. The activity of PTEN is regulated through several molecular
mechanisms. For example, PTEN can be degraded by the ubiquitin proteasome system (UPS), which
is stimulated by neural precursor cell expressed developmentally down-regulated gene 4 (NEDD4)50.
When NEDD4 is deleted branching of axons is inhibited. This effect can be rescued by deletion of
PTEN50. PTEN activity can be stimulated by semaphorin 4D (Sema4D). Sema4D is an axon guidance
molecule for which neurons have a specific receptor; PlexinB1. Binding of Sema4D to PlexinB1
stimulates PTEN activity, which leads to collapse of the growth cone43. Furthermore, PTEN activity
can be opposed by regulating PI3K. Activity of PI3K, which catalyzes the opposite reaction of PTEN
from PIP2 to PIP3 (figure 3), is localized at the tip of the axon51. When PI3K is inhibited, axon
formation is delayed47, which is an effect of PTEN activity. Together, these findings show PTEN
activity can be regulated through several molecular mechanisms. The molecular pathway of PTEN is
crucial for axonal development through the regulation of actin, tubulin and translation, and the
activity of PTEN is controlled by several mechanisms.
Discussion
In this review I have provided an overview of the effects of PTEN deletion, with a focus on the
development of the thalamocortical tract. The goal of this review is to find new starting points for
ASD research. Despite ongoing research, no molecular mechanism based treatments for ASD exist.
Aberrant formation of the thalamocortical neuronal tract is considered an important moment in the
development of the ASD brain9. Many researchers in the ASD field focus on the thalamocortical
axons because the thalamus prioritizes between different kinds of sensitive input when it
communicates to the cortex. Aberrant prioritizing between different forms of sensory input to the
brain causes many of the ASD symptoms. The role of PTEN, a known risk gene for ASD, in the
formation of the thalamocortical axon tract, remains unclear even though many PTEN cKO mouse
lines have been created and much is known about it molecular pathway. This review provided an
overview of the knowledge about the morphology of PTEN cKO mice and the main proteins in its
molecular pathway, in order to provide a hypothesis about the role of PTEN in the formation of the
thalamocortical axon tract. Here, I provide the considerations on which the hypothesis is based.
Subsequently, I formulate the hypothesis and criteria for the investigation of this hypothesis.
Experiments designed on basis of the provided criteria will provide new starting points for the
development of target based treatments for ASD.
10
Based on the overview of the morphological knowledge of PTEN cKO models, it can be
hypothesized that PTEN regulates the formation of the thalamocortical tract. The hypothesis will be
formulated for a cKO model of PTEN in the thalamus, since the development of the thalamocortical
tract starts in the thalamus. Since PTEN deletion leads growing axons to pass their target in
neuromuscular junction formation experiments40, it is hypothesized the axons of the thalamocortical
tract will grow beyond layer four where they usually terminate. Surprisingly, in an optic nerve
regeneration experiment PTEN deletion was part of a combinatory approach to form new effective
axonal tracts39, which negates the new hypothesis that axons will grow past their targets. However,
in this model PTEN was depleted only temporarily, which makes it possible that PTEN expression was
recovered when axonal target binding occurred. Moreover, the finding that the axonal outgrowth
was possible when PTEN was deleted in this model, leads to the hypothesis that development of the
thalamocortical axons will largely follow the route they take in the healthy mouse brain. Apparently,
their initial outgrowth from their cell bodies is less dependent on PTEN as the final formation of the
tips of the axons which occurs later. In conclusion, it is likely PTEN has a function in the inhibition of
the elongation of the axon when the axon has reached the target.
Further development of the hypothesis on the function of PTEN in the development of the
thalamocortical axons can be made. Furthermore, because PTEN will be depleted in the thalamus, it
is likely the overall organization of the thalamus in distinct nuclei will be disrupted. This can be
hypothesized because several cKO mice presented with a disorganized brain region. The disruption of
the nucleus formation in the thalamus, which starts at E14.5 and is morphologically apparent at
E17.552, 53, 53, will probably cause axons to be disorganized when they leave the thalamus. Normally,
the axons leave the thalamus in strictly organized fashion, whereby for example input from one limb
does not mix with input from another limb52. The axons from the VPM and the Po even cause barrel
formation in the cortex, which is also likely to be disrupted in the cKO mouse, leading the axons to
branch more and spread over a wider area. The organization of the axons will probably also be
disrupted because of the loss of adhesion junctions, which was found in retinal progenitors when
they lacked PTEN25. Adhesion junctions are used by cells to communicate to each other and localize
properly. The loss of adhesion junctions will cause axons to fail to recognize each other, which will
cause them to twist amongst each other. The widely spread axons will presumably also form more
synapses with the neurons of the cortex, since PTEN deletion leads to increased synaptogenesis36.
Together, these findings lead to the hypothesis that loss of PTEN in the thalamus will cause the
thalamocortical tracts to twist through each other on their route to the cortex, and at the cortex they
will spread their branches over a wider cortical area and they will form more synapses.
Based on the molecular mechanisms involved in the pathway of PTEN it is likely loss of PTEN
leads to extended growth of the thalamocortical axons. PTEN inhibits the protein pathways which
make it possible the actin filaments and the microtubule are elongated (figure 3). PTEN decreases the
available amount of PIP3, which leads to deactivation of Rac1 and Akt. Downstream of these proteins
regulation of actin and tubulin takes place. PTEN also directly deactivates DBN, which is an actin
binding protein which stimulates the formation of new actin filaments. Moreover, the mTor pathway,
which stimulates protein translation, is inhibited by PTEN. An increase in protein translation
facilitates the formation of new actin and tubulin for the expansion of the cytoskeleton. PTEN can be
stimulated or inhibited through axon guidance molecules. The overview of the molecular
mechanisms in which PTEN is involved, point towards a role for PTEN in the inhibition of the
molecular pathways which organize actin filaments, microtubule and protein translation.
11
Interestingly, a cKO model in the thalamus for tuberous sclerosis 1 (TSC1), a key protein in the PTEN
molecular pathway, was recently researched54. In this model, gastrulation brain homeobox 2 (Gbx2)
was used to target the thalamus. Deletion of TSC1 in the thalamus disrupts thalamic circuitry;
animals showed excess axonal processes in deep cortical layers and an overall increase in thalamic
axons (figure 4)54. Moreover, deletion of TSC1 disturbed the formation of barrels in the cortex. The
barrels were distributed over a larger area and they were less defined. These findings indicate loss of
TSC1 leads to a disruption of the thalamocortical circuitry. Since TSC1 and PTEN are closely related in
the mammalian target of rapamycin (mTOR) regulation pathway (figure 3), it is likely a similar result
will be found with a cKO model for PTEN in the thalamus. PTEN inhibits Akt activity and Akt inhibits
TSC1. Therefore PTEN activity leads to TSC1 activity. For these reasons, a phenotype similar to the
phenotype found in the TSC1 cKO model is likely to be found in a cKO model for PTEN.
Figure 4. TSC1 deletion disrupts thalamocortical circuit formation. Immunohistochemistry was used
on brain slices from control and TSC1 cKO mice. In the cKO mice, TSC1 was deleted from E12. Staining
of thalamocortical axons was performed with RFP and Hoechst staining was used to dye DNA. The
areas in which the pictures on the right were taken are delineated in the left pictures. In the TSC1
cKO mice barrels are diffuse compared to the ones in control mice. The TSC1 cKO mice also had
excess axonal processes in the deep cortical layers (arrow)54.
Based on the insights gathered in this review it is likely that PTEN regulates axon tract formation by
slowing down axon growth, once the axon is at its target. Furthermore, PTEN regulates the
orientation of the axons to the right barrel of the cortex. Presumably, PTEN deletion causes
thalamocortical axons to grow beyond their targets, divide more amongst barrels and form more
synapses (figure 5). In order to investigate this hypothesis a cKO model of PTEN in the thalamus
should be studied. Morphological effects should be found, such as a disruption of the barrel
formation in the cortex, an increase in the amount of axons and deeper growth of the axons into the
cortex. Another criterion for this hypothesis is the presence of downstream effects in the molecular
pathway of PTEN. Increased activity of proteins such as Rac1, Akt, mTor and CRMP2 is to be
expected. A final validation method for this hypothesis is the potential of a rescue experiment.
Presumably, the morphological and molecular effects of the deletion of PTEN can be rescued by
inhibiting PI3K. PI3K catalyzes the reaction from PIP2 to PIP3, and thereby antagonizes PTEN.
Therefore, simultaneous deletion of PTEN and PI3K should lead to normalization of the phenotype. In
conclusion, PTEN is hypothesized to have a crucial role in organizing the development of the
thalamocortical axons and allowing them to slow down at their target to form correct synapses.
12
A
B
Figure 5. Hypothesis on axon growth in WT mice and PTEN cKO mice. (A) In WT mice
thalamocortical axons grow through the subplate into layer four in of the cortical plate. (B) In PTEN
cKO mice the thalamocortical axons spread more broadly and grow further into the marginal zone.
Experiments with cKO models of PTEN have provided insight in the important function of PTEN in
brain development. PTEN regulates proliferation and migration of neurons. However, though it is
clear that PTEN is crucial for axonal tract formation, the exact function of PTEN and the molecular
pathways involved remain unknown. Based on the overview provided in this review, a hypothesis
about the involvement of PTEN in the formation of the thalamocortical tract was formulated: PTEN
slows the thalamocortical axons down when they reach their target to allow for synaptogenesis in
the correct location. Furthermore, PTEN regulates nucleus formation in the thalamus, which makes it
possible for the thalamocortical tract to be correctly organized and cause barrelization in the
somatosensory cortex. PTEN influences the formation of the thalamocortical tract through the
dephosphorylation of PIP3 to PIP2. To test this hypothesis, experiments with a cKO model of PTEN in
the thalamus are necessary. Such experiments will allow for the identification of the precise
pathways and morphological effects of PTEN activity. Moreover, the proposed rescue experiments
with inhibitors of the PTEN antagonist PI3K may provide a starting point for the development of new
treatments for ASD. In conclusion, PTEN is an important regulator of thalamocortical tract formation
and it is a promising protein to gather new insights in the ASD brain.
13
Lay summary
Worldwide 1% of the population suffers from autism spectrum disorder (ASD), but treatments to
sufficiently suppress the symptoms do not exist. One of the key symptoms is the failure to prioritize
between observations, which causes patients to fail to function in environments with many stimuli.
This key symptom is thought to be due to a disruption of the communication between the thalamus
and the cortex. The cortex is the part of our brain which is responsible for all higher order functioning
and it is the role of the thalamus to order and prioritize the input the brain receives from the
peripheral nervous system for the cortex, before the input reaches the cortex. For this reason, the
formation of the axon tracts which carry the information from the thalamus to the cortex is one of
the main focuses of ASD research. In this review, a hypothesis on the role of PTEN in the formation of
these axon tracts is presented and proposals for the investigation of this hypothesis are made. The
hypothesis is based on the current knowledge on PTEN, which is known to be a risk factor for ASD.
PTEN is important to slow down cell replication and growth. The new hypothesis offered in this
review is that PTEN is necessary to slow axons down when they have reached their target in order to
prevent them from passing their target. Furthermore, PTEN presumably organizes the development
of the thalamus, and therefore PTEN is also necessary to organize the order in which the axons grow
from the thalamus to the cortex. Several experiments to test this hypothesis are proposed as well as
a potential pharmacological target to rescue phenotypes in mice which lack PTEN. Hopefully the
proposed experiments will one day lead to the development of new medication for autism spectrum
disorder.
Acknowledgements
I thank Kristel Kleijer for her supervision of the writing of this review.
14
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