1.0 number of cases of neurodegenerative disorders, especially

1.0 IntroductionAS1 


The long-term aspiration of regenerative medicine is to
stimulate mechanisms in humans that could lead to the functional repair and/or
replacement of lost or damaged tissues, organs. This aspiration also includes the
treatment of a number of age related neurodegenerative disorders that affect
our normal daily life such as Parkinson’s and Alzheimer’s disease. Current
therapies available for neurodegenerative disorders may improve the quality of
life but do not cure the disease. There is an increase in number of cases of
neurodegenerative disorders, especially in developed countries, which creates
both social and economic burden and highlights a need to find novel therapies.

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Interestingly, within the vertebrates, certain taxa, especially urodele
amphibians (salamanders), which includes newts shows wide spread regenerative capabilities.

Newts, have been extensively studied for their regenerative potential,
including their central nervous system.


Previously, studies from our laboratory showed that in
contrast to mammals, newts are able to regenerate dopamine neurons in the adult
midbrain. Midbrain dopamine neurons are particularly interesting because their
degeneration is the major hallmark of Parkinson’s disease. Dopaminergic
regeneration in newts proceeds by quiescent ependymoglia cells reentering the
cell-cycle as a response to neuronal ablation.

Notably, after completion of the regenerative events, are
regenerated these ependymoglia cells returned to quiescence. Interestingly,
under certain disease conditions mammalian neural stem cells (NSCs) also respond
to injury by activation but this process does not lead to the production of
significant number and functional integration of new neurons. It is important
however to point out that evolutionary similarities between newt ependymoglia
cells and mammalian NSCs do exist. For example, based on findings in newts,
which showed that proliferation of midbrain ependymoglia cells were under the
control of dopamine signalling, our lab was able to increase dopamine
neurogenesis in mice (Hedlund et al, 2016, Sci Rep). These data support the
view that studies on newts could provide clues to how to manipulate the mammalian
brain to improve recovery in neurodegenerative disorders.


Since newt ependymoglia cells play a central role in
neuronal regeneration, it is important to gain mechanistic understanding of
their unique regenerative potential. Therefore, this thesis focusses on
detailed characterisation of ependymoglia cells in newts both in the adult
brain as well as during their maturation in a developmental context.  Understanding the developmental origin of ependymoglia
cells, how they mature, acquire quiescence and comparing them to their
mammalian counterparts might reveal critical interspecies differences.


In this thesis, efforts were made for a detailed
characterisation of ependymoglia cells from their developmental origin to adult
stage to understand their unique nature. 
In the introductory part of this thesis, I give a general overview of
the field of regeneration biology with emphasis on regenerative ability in
salamanders. The maturation of neural progenitors from embryonic to adulthood
and adult NSCs potential to respond to injury are discussed in the following
section. Third, I discuss the role of reactive oxygen species in neurogenesis, including
evolutionary considerations. In the last part I summarise the findings of the
papers included in this thesis.






























1.1 Historical
overview of regeneration:


The concept of regeneration has fascinated the scientists for
centuries. One of the earliest dated account on regeneration originates from
Greek mythology, where Prometheus – the half god half man- was punished by Zeus
for disobeying God’s order and gave fire to mankind, an act of disobedience. Prometheus
was chained to a rock and an eagle peck his liver every day. The lost part of
his liver grew back each night. Myths are bit exaggerated and in reality, it is
not possible to regenerate the liver overnight. However, scientific evidence prove
that liver of humans can partially regenerate (Ingle D.J et al1957, Chen M.E et
al 1991). Another mythology came from the tale of three hags in the legend of
Mercury, where the concept of eye regeneration was coined. Where hags had only
one eye among them, if another hag wanted to see, they took the eye ball from
an orbit and passed it to another hags orbit. The story is indeed mythical,
however, experimental manipulation showed that the removal of an eyeball from
newts and grafting it back to the visual orbit leads visual recovery (Stone et
al 1963). Also, now we know that newts have remarkable ability to regenerate
the lens repeatedly without any sign of age-related decline (Tsonis P.A et al
2004). Apart from the Greek mythological beliefs, first scientific discoveries
of regeneration were documented by Aristotle (384-322BC), in his book “The
history of Animals”, he mentioned about tail regeneration of lizards, however
until 18th century there was no scientific report about the
regeneration abilities in animals.


The first report on
regeneration based on experimental evidences dates back to 1712, where, the
French scientist Rene-Antonio Ferchault de Reaumur (1683-1757), published a
paper about regeneration of the legs in fresh water grayfish. Reaumur noted that when he frequently attempted to cut the portion of
the limb, which led to autotomy, and the limb regenerate rapidly when cut at
the autotomy plane than anywhere else. This indicated that the region which
prone to injury could regenerate rapidly. Reaumur was also curious about the
origin of the limb regeneration. Except the sac attached to the injured area,
there was no external visibility of limb. Therefore, he dissected out the sac
and found out that limb is indeed growing within the sac.  Later that century, another breakthrough occurred
in the field of regenerative biology. Abraham Trembley (1710-1784), a Swiss naturalist had
discovered regeneration in the polyp, hydra. When he first looked at polyp, he
had curiosity whether it is an animal or a plant. When he noted that it has
step-by-step movement, he predicted it to be an animal. His curiosity for
regeneration, came by when he noticed that not all polyps have similar number
of arms. Trembley coined the term hydra, after cutting the hydra repeatedly and
seen seven headed polyps. Which looked like a monster, from Greek mythology


The earliest studies on regeneration of vertebrates came
from Italaian Scientist Lazzaro Spallanzani (1729-1799). In 1768, he looked at
pre-metamorphic frogs and toads, and given an explanation that they could
regenerate the tail. Spallanzani is the first to describe regeneration of limbs
in salamanders after amputation. He documented the appearance of small round
stump at the injury site, now this structure is called blastema and critical
for limb regeneration to progress (Bryant S.V et al 2002). Spallanzani also recorded
tail regeneration in the newts.


The discovery that certain adult vertebrates could
regenerate led to several studies on the regenerative abilities in adult
vertebrate species, and also led to a speculation of why only certain species
have regenerative potential. Thomas Hunt Morgan (1866-1945), a renowned
geneticist and August Weismann (1834-1914) known for his famous ‘germ plasm
theory’, both had different view on animals’ ability to regenerate. Weissmann
believed that regeneration is adapted to species, and organs which are prone to
injury have evolved the regenerative potential independently.  However, Morgan was against this theory, he
argued that if regeneration occurs to species which are prone to injury, then
how about species/organs which are not prone to injury?  Morgan tried to explain this theory by
amputating salamander and crab legs, where no natural injury occurs and proved
they do regenerate. He considered that regeneration is innate to certain
species and might have lost in other species (Morgan T.H 1901). Irrespective of
their claims, still there is ongoing debate about whether regenerative ability
is inherited or adapted. I will discuss this view in detail towards the
concluding chapter in the thesis.












T.H Morgan in 1901, in his book Regeneration classified regeneration into Morphallaxis and
Epimorphosis based on whether regeneration require proliferating cells or not.



Morphallaxis, where regeneration does not require active
proliferating cells, animals could regenerate by active remodelling of existing
tissues. Morgan from his studies on planarian regeneration concluded that
planarian regeneration occurs by tissue remodelling (Morgan T.H et al 1898). He
came to this conclusion after monitoring how planarians regenerate from 1/279th
of tissue. But recent evidence indicates that planarian regeneration occurs by
stem cells called neoblast which are the only proliferating cells in planarians
(Reddien P.W et al 2004, Wagner D.E et al 2011). Apart from planarian, hydra
regeneration also thought to be mediated by tissue re-organisation. Inhibiting
DNA synthesis via hydroxy-urea showed that regeneration is independent of
mitosis (Cummings S.G et al 1984). However, recent evidences show that indeed
proliferating cells, which looks like blastema under apoptotic cells after
injury is important for hydra regeneration (Chera S et al 2009). Therefore,
species thought to regenerate by morphallaxis, still require proliferating
cells for their regeneration. Morphallaxis is an old term, currently no known
species which regenerate without requirement of proliferating cells.



The most accepted model where there is a requirement of
proliferating cells to regenerate the lost tissues. The requirement of
proliferating cells has been demonstrated in hydra and planarians as well as
among different vertebrates including newts. Among the animals examined so far
for their regenerative mechanisms, the regeneration occurs through
dedifferentiation and transdifferentiation of matured somatic cells as well as
stem cells in the adult tissues.


Transdifferentiation is the process where one matured cell
is converted to another cell type without any intermediate stage. In a
regeneration context, transdifferentiation has been conclusively demonstrated
during lens regeneration in newts. In newts, only the dorsal iris retains
regenerative potential, and upon lens removal, pigment epithelial cells (PECs)
change morphology, proliferate and transdifferentiated into lentoid bodies to
form the new lens. (Eguchi G et al 1993, Henry J.J et al 2010).

Transdifferentiation is not a predominant source of regeneration in other tissues.


Dedifferentiation is the process where terminally
differentiated cells dedifferentiate to an intermediate stage before their
redifferentiation. Newt limb regeneration is a typical example of
dedifferentiation. Upon injury, the multinucleated muscle fibers, fragment to
produce mononucleated cells and these mononucleated cells proliferate and
contribute to blastemal formation, which leads to regeneration of the limb (Lo
D.C et al 1993, Echeverri K et al 2001, Wang H et al 2015). Apart from newts,
zebrafish regenerates their heart upon injury, and recent experiments
demonstrate that this event are also mediated by dedifferentiation of
cardiomyocytes. Lineage tracing of differentiated cardiomyocytes indicates that
after injury cardiomyocytes dedifferentiation and reenter cell cycle which
contribute to regeneration (Jopling C et al 2010). Regenerative potential
exists in mouse neonatal heart, and lineage tracing studies indicate the
possibility of cardiac myocytes contributing to regeneration by dedifferentiation
(Porrello E.R et al 2011). However, adult mice lack regenerative ability of the
heart tissues.


Proliferating adult stem cells in organisms also contribute
to regeneration. The neoblast in adult planarians are critical for their
regeneration, upon injury they generate all major cell types needed for
regeneration. If irradiated, then they lose the neoblast and eventually die,
and transplantation of single neoblast to irradiated host is sufficient for
their regeneration (Wagner et al 2011).  Newt
limb regeneration is also mediated by activation of satellite cells, which
reenter cell cycle and contribute to functional regeneration after injury
(Morrison J.I et al 2006). Zebrafish and newt brain regeneration is also
mediated by activation of stem cells present in the brain (Berg et al 2011,
Kizil C 2012).


From our current understanding with all the organisms which
retain regenerative potential, it appears that there is a pre-requirement of
proliferating cells to regenerate and replace damaged tissues. Interestingly,
there are species which retain proliferating adult stem cells, still they have
very restricted ability to regenerate.




1.3 Habitat of


Amphibians which retain tail after metamorphosis are called
urodele amphibians, which include newts and salamanders. The common newts used
in regeneration research are Cynops
pyrrhogaster (Japaneese fire-bellied newts), Notophthalmus Viridescens (Eastern spotted newts) and Pleurodeles waltl (Iberian ribbed newts) (Ueda
Y et al 2005 Berg A et al 2010, Joven A et al 2016).


The eastern spotted newts are found throughout North-Eastern
America. Newts have a complex life cycle, which is categorised to larval, eft
and adulthood (Brockes J et al 2005, Joven A et al 2016). However, in Iberian
newts the larval stages are more complex and divided into initial, early active
and late active larva stages (Joven A et al 2016). Each stage is specified by a
set of external changes and acquisition of complex behaviours. The newt larvae
mainly use gills and skin for their oxygen uptake and do not have lungs.

However, during larval metamorphosis, they lose their gills and develop lungs,
which is essential for them in postmetamorphic stages to on move to terrestrial
life (Shi D et al 1995).  During metamorphosis,
a number of external changes occurs in newts. In Eastern spotted newts, this includes changes in the anatomical organisation of the skin as well as colour
to a reddish tone and called efts and
live in a terrestrial habitat for about one to three years. After this terrestrial
stage, they return to water and the skin colour changes to greenish-grey
(Brockes J et al 2005). Adult spotted newts prefer to breed and lay eggs in
water, even during winter under an ice-covered pond (Berner N.J et al 2010). In
the wild, the life span of spotted newts is up to 15 years (Hillman et al


The Iberian ribbed newts are wide-spread from Iberian
Peninsula to Morocco. Similar to Eastern spotted newts they also have complex
life style and undergo all three stages (Joven A et al 2016). They are more aquatic than Eastern
spotted newts and grow up to 30 cm in size.

The advantages of the Iberian
ribbed newts as a laboratory model is ease of breeding in captivity,
availability of large number eggs where a single female lay about 200 eggs at a time (Teunis B et al 2005), and transgenesis (Joven A et al 2016, Elewa A et al 2017).

Irrespective of their variation in habitat and breeding, both Eastern spotted
newts and Iberian ribbed newts retain wide-spread regenerative capacity and
comparing inter-species regenerative ability will help us understand
regeneration in an evolutionary perspective.


1.4 Regeneration
in newts


As discussed earlier, the first report on the regenerative
ability in newts’ dates back to Lazzao Spallazani in early eighteenth century.

Spallazani described regeneration of limbs and tail in newts. Experiments
spanning the 20th century have shown that the newts regenerate almost all body
parts and they are called the champions of regeneration. Newt has the potential
to regenerate lens, jaws, heart, tail, and limb (Brockes J.P et al 1997, Iten
L.E et al 1976, Davis et al 1990, Tsonis P.A et al 2004).

Interestingly, not only newt possess wider spectrum of
regenerative ability, it is also noted there is no sign of age related decline
in their regenerative ability. Newts can regenerate lens, and removing lens
repeatedly for 18 times on the same animal revealed regenerate of lens not
altered by repeated injury. (Eguchi G et al 2011).  


Apart from the appendages, newts also possess ability to
regenerate injured CNS, spinal cord and brain. Spinal cord regeneration has
been extensively studied in newts. Most of the spinal cord studies have been
done after amputation of the tail. However, transection of spinal cord proximal
to the hind limb was performed to study the regenerative ability and behaviour
response. In this context, the newts were able to regenerate the spine and
recovered hind limb movement by four weeks after spinal cord injury (Davis et
al 1990). Spinal cord injury was also performed to assess the axonal
regeneration contribute of cells to regeneration (Zukor et al 2011). The
paedomorphic salamander, axolotl, has also been extensively studied for their
spinal cord regeneration. These experiments indicate that neural stem cell-mediated
proliferation contribute to spinal cord regeneration (Albors A.D et al 2015).


Adult newts are able to regenerate parts of the brain after
mechanical lesioning. In classical experiments in amphibians the approach was
to remove the optic tectum and study their functional outcome. In newts,
removal of optic tectum and assessment of the brain till 90 days indicates that
they are able to regenerate the optic tectum (Minneli G et al 1987). In another
study, the retinotectal projection pathway was analysed after partial optic
tectum removal. This study has revealed that the newts regenerate the optic tectum
and most of the retinotectal projection were recovered by 8 months (Okomoto M
et al 2007). Recently, number of studies on brain regeneration has been
performed on newts and this will be discussed later in this chapter.


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