4 research outputs found
Cytoskeleton-interacting proteins in brainstem development : Roles of KCC2 and Vangl2
The brainstem is the most evolutionary conserved division of the brain. It develops from the
hindbrain and midbrain regions of the neural tube and forms neural networks that regulate
vital functions of the body. One of the most critical roles is to generate respiratory rhythm
for the regulation of oxygen, carbon dioxide and pH levels. This is achieved by pacemaker
neurons and neural networks in the medulla oblongata, controlled by different modulatory
systems. The mechanisms whereby the respiratory rhythm is generated and regulated are not
fully understood and have only recently started to be unveiled.
This thesis describes the importance of two different gene products, KCC2 and
Vangl2, for proper development of the brainstem. We show that, while these genes act in
separate phases of development, they share the common feature of regulating the integrity of
the neuronal cytoskeleton necessary for maturation of the brainstem.
KCC2 is a neuronal K+/Cl- cotransporter that is responsible for the developmental
shift in the postsynaptic response to GABA. A fundamental premise for this thesis is that we
found KCC2 protein expression in the hindbrain region of mice already at embryonic day
9.5, although its ion transport activity does not become functional until late fetal age. We
show that the depolarizing effect of GABA elicits increased activity of fetal respirationrelated
neurons. In addition, the developmental GABA shift is associated with plasma
membrane targeting of KCC2 in respiration-related regions of rats around birth.
Overexpression of KCC2 in the mouse neural tube resulted in altered neuronal
differentiation and neural crest migration. These effects were independent of the ion
transport function of KCC2 and were shown to rely on a structural interaction with the
cytoskeleton-associated protein 4.1N. Thus, transport-inactive KCC2 may regulate neuronal
differentiation and migration during early development. We assessed the early importance
of KCC2 further in mice knockout for this gene, which die at birth from respiratory failure.
Brainstem organotypic cultures of these mice displayed a lower correlated network activity
in the preBötzinger region. In addition, characterization of the respiration-related regions
showed less glutamatergic synapses in the parafacial respiratory group of KCC2-deficient
mice. This indicates that KCC2 is essential for the maturation of respiratory neural
networks.
Finally, we show that the planar cell polarity gene Vangl2 regulates neural tube
closure in the hindbrain region by promoting the formation of adherens junctions. Vangl2
was found to structurally interact with Rac1. Moreover, disruption of adherens junctions by
a partial blockade of Rac1 could be rescued by Vangl2. This suggests that Vangl2 plays a
critical role in the recruitment of Rac1 to the adherens junctions.
In conclusion, the results presented in this thesis increase our knowledge of
brainstem development, from closure of the neural tube until the formation of functional
neural networks. Our findings have potential implications for research and understanding of
neural tube defects as well as breathing disorders, such as congenital central hypoventilation
syndrome, that arise from aberrant formation of the neural networks constituting the central
pattern generator for breathing
CO2-evoked release of PGE2 modulates sighs and inspiration as demonstrated in brainstem organotypic culture
Inflammation-induced release of prostaglandin E-2 (PGE(2)) changes breathing patterns and the response to CO2 levels. This may have fatal consequences in newborn babies and result in sudden infant death. To elucidate the underlying mechanisms, we present a novel breathing brainstem organotypic culture that generates rhythmic neural network and motor activity for 3 weeks. We show that increased CO2 elicits a gap junction-dependent release of PGE(2). This alters neural network activity in the preBotzinger rhythm-generating complex and in the chemosensitive brainstem respiratory regions, thereby increasing sigh frequency and the depth of inspiration. We used mice lacking eicosanoid prostanoid 3 receptors (EP3R), breathing brainstem organotypic slices and optogenetic inhibition of EP3R(+/+) cells to demonstrate that the EP3R is important for the ventilatory response to hypercapnia. Our study identifies a novel pathway linking the inflammatory and respiratory systems, with implications for inspiration and sighs throughout life, and the ability to autoresuscitate when breathing fails.Peer reviewe
Early function of KCC2 and wnt genes : Cytoskeletal effects in neural stem cells
From closure of the neural tube until formation of neuronal networks,
cells undergo several crucial changes which depend on the expression of
many important molecules. GABA, the principal inhibitory neurotransmitter
in the adult nervous system, acts as an excitatory signal during
embryonic development. This is a necessary neurotrophic signal resulting
from a high level of chloride in neural cells. During neural maturation,
the potassium-chloride co-transporter, KCC2, lowers the intracellular
chloride level and switches the GABA response to hyperpolarizing. Studies
have shown a role for KCC2 in the formation of neuronal dendrites but
earlier functions in mammals have not yet been revealed. Among the most
well-known factors regulating early nervous system development are the
Wnt proteins, which act as extracellular ligands. This thesis describes
the action of KCC2 and Wnt genes on the cytoskeleton of neural
progenitors and demonstrates how a change in their expression can alter
neural cell behaviour.
To correlate the expression pattern of KCC2 with functionality, we
recorded the GABA response in fetal rat respiratory neurons. The
developmental switch from depolarizing to hyperpolarizing was shown to be
around embryonic day (E) 20. The KCC2 expression pattern in the main
respiratory-related nuclei preBötzinger complex and parafacial
respiratory group changed drastically between E18 and P0. From being
essentially cytoplasmic at E18, KCC2 appeared to be translocated to the
plasma membrane at E20. At P0, the KCC2 protein was localized
predominantly at the plasma membrane and maintained this expression
pattern postnatally.
Overexpression of KCC2 in the neural tube of transgenic mouse embryos had
a deleterious effect on the nervous system development. Transgenic
embryos at E9.5-E13.5 displayed a reduced neuronal differentiation and
impaired neural crest migration. Similar results were obtained with a
truncated form of KCC2, lacking the sequence for ion transport, implying
that the effects were not GABA-dependent. Interestingly, the neural tube
of transgenic embryos had an aberrant distribution of the cytoskeletal
protein actin, suggesting an interaction between KCC2 and the
cytoskeleton.
In a related study, we examined transgenic embryos with neural-specific
overexpression of Wnt7a. These embryos have been shown to exhibit
cytoskeletal defects discernible as impaired adherens junctions in the
rostral neural tube. Analysis of E9.5 and E10.5 transgenic embryos showed
a reduced neuronal differentiation, indicating a role for Wnt7a in the
control of neuronal progenitor maturation. Moreover, the downstream
signalling gene Vangl2 was upregulated in the neural tube. We therefore
studied transgenic embryos overexpressing Vangl2 in neural progenitors
and loop-tail embryos with a natural mutation in the Vangl2 gene. Both
Vangl2 gain-of-function and loss-of-function resulted in cytoskeletal
defects in the neural tube, characterized by aberrant distribution of
actin and other cytoskeletal components. Moreover, the small GTPase Rac1
was redistributed in the cells of the neural tube, indicating an
interaction between Vangl2 and Rac1. Cell studies using HEK293T, MDCK and
C17.2 cell lines showed similar effects of Vangl2 on the cytoskeleton as
well as altered cell adhesion and motility. Interestingly, these effects
could be blocked by a Rac1 knock-down, verifying the interaction between
Vangl2 and Rac1 observed in vivo. Taken together, these results
demonstrate the significance of a coordinated expression of
cytoskeletal-interacting proteins during nervous system development