1.0 number of cases of neurodegenerative disorders, especially

1.0 IntroductionAS1  The long-term aspiration of regenerative medicine is tostimulate mechanisms in humans that could lead to the functional repair and/orreplacement of lost or damaged tissues, organs.

This aspiration also includes thetreatment of a number of age related neurodegenerative disorders that affectour normal daily life such as Parkinson’s and Alzheimer’s disease. Currenttherapies available for neurodegenerative disorders may improve the quality oflife but do not cure the disease. There is an increase in number of cases ofneurodegenerative disorders, especially in developed countries, which createsboth social and economic burden and highlights a need to find novel therapies.Interestingly, within the vertebrates, certain taxa, especially urodeleamphibians (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 incontrast to mammals, newts are able to regenerate dopamine neurons in the adultmidbrain.

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Midbrain dopamine neurons are particularly interesting because theirdegeneration is the major hallmark of Parkinson’s disease. Dopaminergicregeneration in newts proceeds by quiescent ependymoglia cells reentering thecell-cycle as a response to neuronal ablation. Notably, after completion of the regenerative events, areregenerated these ependymoglia cells returned to quiescence. Interestingly,under certain disease conditions mammalian neural stem cells (NSCs) also respondto injury by activation but this process does not lead to the production ofsignificant number and functional integration of new neurons. It is importanthowever to point out that evolutionary similarities between newt ependymogliacells and mammalian NSCs do exist.

For example, based on findings in newts,which showed that proliferation of midbrain ependymoglia cells were under thecontrol of dopamine signalling, our lab was able to increase dopamineneurogenesis in mice (Hedlund et al, 2016, Sci Rep). These data support theview that studies on newts could provide clues to how to manipulate the mammalianbrain to improve recovery in neurodegenerative disorders. Since newt ependymoglia cells play a central role inneuronal regeneration, it is important to gain mechanistic understanding oftheir unique regenerative potential. Therefore, this thesis focusses ondetailed characterisation of ependymoglia cells in newts both in the adultbrain as well as during their maturation in a developmental context.

  Understanding the developmental origin of ependymogliacells, how they mature, acquire quiescence and comparing them to theirmammalian counterparts might reveal critical interspecies differences. In this thesis, efforts were made for a detailedcharacterisation of ependymoglia cells from their developmental origin to adultstage to understand their unique nature. In the introductory part of this thesis, I give a general overview ofthe field of regeneration biology with emphasis on regenerative ability insalamanders. The maturation of neural progenitors from embryonic to adulthoodand adult NSCs potential to respond to injury are discussed in the followingsection. Third, I discuss the role of reactive oxygen species in neurogenesis, includingevolutionary considerations. In the last part I summarise the findings of thepapers included in this thesis.                             1.

1 Historicaloverview of regeneration: The concept of regeneration has fascinated the scientists forcenturies. One of the earliest dated account on regeneration originates fromGreek mythology, where Prometheus – the half god half man- was punished by Zeusfor disobeying God’s order and gave fire to mankind, an act of disobedience. Prometheuswas chained to a rock and an eagle peck his liver every day. The lost part ofhis liver grew back each night. Myths are bit exaggerated and in reality, it isnot possible to regenerate the liver overnight. However, scientific evidence provethat liver of humans can partially regenerate (Ingle D.J et al1957, Chen M.

E etal 1991). Another mythology came from the tale of three hags in the legend ofMercury, where the concept of eye regeneration was coined. Where hags had onlyone eye among them, if another hag wanted to see, they took the eye ball froman orbit and passed it to another hags orbit. The story is indeed mythical,however, experimental manipulation showed that the removal of an eyeball fromnewts and grafting it back to the visual orbit leads visual recovery (Stone etal 1963). Also, now we know that newts have remarkable ability to regeneratethe lens repeatedly without any sign of age-related decline (Tsonis P.A et al2004). Apart from the Greek mythological beliefs, first scientific discoveriesof regeneration were documented by Aristotle (384-322BC), in his book “Thehistory of Animals”, he mentioned about tail regeneration of lizards, howeveruntil 18th century there was no scientific report about theregeneration abilities in animals. The first report onregeneration based on experimental evidences dates back to 1712, where, theFrench scientist Rene-Antonio Ferchault de Reaumur (1683-1757), published apaper about regeneration of the legs in fresh water grayfish.

Reaumur noted that when he frequently attempted to cut the portion ofthe limb, which led to autotomy, and the limb regenerate rapidly when cut atthe autotomy plane than anywhere else. This indicated that the region whichprone to injury could regenerate rapidly. Reaumur was also curious about theorigin of the limb regeneration. Except the sac attached to the injured area,there was no external visibility of limb.

Therefore, he dissected out the sacand found out that limb is indeed growing within the sac.  Later that century, another breakthrough occurredin the field of regenerative biology. Abraham Trembley (1710-1784), a Swiss naturalist haddiscovered regeneration in the polyp, hydra. When he first looked at polyp, hehad curiosity whether it is an animal or a plant. When he noted that it hasstep-by-step movement, he predicted it to be an animal. His curiosity forregeneration, came by when he noticed that not all polyps have similar numberof arms.

Trembley coined the term hydra, after cutting the hydra repeatedly andseen seven headed polyps. Which looked like a monster, from Greek mythologyhydra.   The earliest studies on regeneration of vertebrates camefrom Italaian Scientist Lazzaro Spallanzani (1729-1799).

In 1768, he looked atpre-metamorphic frogs and toads, and given an explanation that they couldregenerate the tail. Spallanzani is the first to describe regeneration of limbsin salamanders after amputation. He documented the appearance of small roundstump at the injury site, now this structure is called blastema and criticalfor limb regeneration to progress (Bryant S.V et al 2002). Spallanzani also recordedtail regeneration in the newts.  The discovery that certain adult vertebrates couldregenerate led to several studies on the regenerative abilities in adultvertebrate species, and also led to a speculation of why only certain specieshave regenerative potential. Thomas Hunt Morgan (1866-1945), a renownedgeneticist and August Weismann (1834-1914) known for his famous ‘germ plasmtheory’, both had different view on animals’ ability to regenerate. Weissmannbelieved that regeneration is adapted to species, and organs which are prone toinjury have evolved the regenerative potential independently.

  However, Morgan was against this theory, heargued that if regeneration occurs to species which are prone to injury, thenhow about species/organs which are not prone to injury?  Morgan tried to explain this theory byamputating salamander and crab legs, where no natural injury occurs and provedthey do regenerate. He considered that regeneration is innate to certainspecies and might have lost in other species (Morgan T.H 1901). Irrespective oftheir claims, still there is ongoing debate about whether regenerative abilityis inherited or adapted. I will discuss this view in detail towards theconcluding chapter in the thesis.

         1.2Regeneration:  T.H Morgan in 1901, in his book Regeneration classified regeneration into Morphallaxis andEpimorphosis based on whether regeneration require proliferating cells or not.  1.2.1MorphallaxisMorphallaxis, where regeneration does not require activeproliferating cells, animals could regenerate by active remodelling of existingtissues. Morgan from his studies on planarian regeneration concluded thatplanarian regeneration occurs by tissue remodelling (Morgan T.H et al 1898).

Hecame to this conclusion after monitoring how planarians regenerate from 1/279thof tissue. But recent evidence indicates that planarian regeneration occurs bystem 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, hydraregeneration also thought to be mediated by tissue re-organisation. InhibitingDNA synthesis via hydroxy-urea showed that regeneration is independent ofmitosis (Cummings S.

G et al 1984). However, recent evidences show that indeedproliferating cells, which looks like blastema under apoptotic cells afterinjury is important for hydra regeneration (Chera S et al 2009). Therefore,species thought to regenerate by morphallaxis, still require proliferatingcells for their regeneration. Morphallaxis is an old term, currently no knownspecies which regenerate without requirement of proliferating cells.

 1.2.2EpimorphosisThe most accepted model where there is a requirement ofproliferating cells to regenerate the lost tissues. The requirement ofproliferating cells has been demonstrated in hydra and planarians as well asamong different vertebrates including newts. Among the animals examined so farfor their regenerative mechanisms, the regeneration occurs throughdedifferentiation and transdifferentiation of matured somatic cells as well asstem cells in the adult tissues.

 Transdifferentiation is the process where one matured cellis converted to another cell type without any intermediate stage. In aregeneration context, transdifferentiation has been conclusively demonstratedduring lens regeneration in newts. In newts, only the dorsal iris retainsregenerative potential, and upon lens removal, pigment epithelial cells (PECs)change morphology, proliferate and transdifferentiated into lentoid bodies toform 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 terminallydifferentiated cells dedifferentiate to an intermediate stage before theirredifferentiation. Newt limb regeneration is a typical example ofdedifferentiation. Upon injury, the multinucleated muscle fibers, fragment toproduce mononucleated cells and these mononucleated cells proliferate andcontribute to blastemal formation, which leads to regeneration of the limb (LoD.

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 experimentsdemonstrate that this event are also mediated by dedifferentiation ofcardiomyocytes. Lineage tracing of differentiated cardiomyocytes indicates thatafter injury cardiomyocytes dedifferentiation and reenter cell cycle whichcontribute to regeneration (Jopling C et al 2010). Regenerative potentialexists in mouse neonatal heart, and lineage tracing studies indicate thepossibility of cardiac myocytes contributing to regeneration by dedifferentiation(Porrello E.R et al 2011). However, adult mice lack regenerative ability of theheart tissues.

 Proliferating adult stem cells in organisms also contributeto regeneration. The neoblast in adult planarians are critical for theirregeneration, upon injury they generate all major cell types needed forregeneration. If irradiated, then they lose the neoblast and eventually die,and transplantation of single neoblast to irradiated host is sufficient fortheir regeneration (Wagner et al 2011).  Newtlimb regeneration is also mediated by activation of satellite cells, whichreenter cell cycle and contribute to functional regeneration after injury(Morrison J.I et al 2006). Zebrafish and newt brain regeneration is alsomediated by activation of stem cells present in the brain (Berg et al 2011,Kizil C 2012). From our current understanding with all the organisms whichretain regenerative potential, it appears that there is a pre-requirement ofproliferating cells to regenerate and replace damaged tissues.

Interestingly,there are species which retain proliferating adult stem cells, still they havevery restricted ability to regenerate.    1.3 Habitat ofnewts Amphibians which retain tail after metamorphosis are calledurodele amphibians, which include newts and salamanders. The common newts usedin regeneration research are Cynopspyrrhogaster (Japaneese fire-bellied newts), Notophthalmus Viridescens (Eastern spotted newts) and Pleurodeles waltl (Iberian ribbed newts) (UedaY et al 2005 Berg A et al 2010, Joven A et al 2016).  The eastern spotted newts are found throughout North-EasternAmerica. Newts have a complex life cycle, which is categorised to larval, eftand adulthood (Brockes J et al 2005, Joven A et al 2016).

However, in Iberiannewts the larval stages are more complex and divided into initial, early activeand late active larva stages (Joven A et al 2016). Each stage is specified by aset of external changes and acquisition of complex behaviours. The newt larvaemainly 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 terrestriallife (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 colourto a reddish tone and called efts andlive in a terrestrial habitat for about one to three years. After this terrestrialstage, 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 inwater, even during winter under an ice-covered pond (Berner N.J et al 2010). Inthe wild, the life span of spotted newts is up to 15 years (Hillman et al2009).  The Iberian ribbed newts are wide-spread from IberianPeninsula to Morocco. Similar to Eastern spotted newts they also have complexlife style and undergo all three stages (Joven A et al 2016). They are more aquatic than Easternspotted newts and grow up to 30 cm in size.The advantages of the Iberianribbed 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 spottednewts and Iberian ribbed newts retain wide-spread regenerative capacity andcomparing inter-species regenerative ability will help us understandregeneration in an evolutionary perspective.

 1.4 Regenerationin newts As discussed earlier, the first report on the regenerativeability in newts’ dates back to Lazzao Spallazani in early eighteenth century.Spallazani described regeneration of limbs and tail in newts. Experimentsspanning the 20th century have shown that the newts regenerate almost all bodyparts and they are called the champions of regeneration. Newt has the potentialto regenerate lens, jaws, heart, tail, and limb (Brockes J.

P et al 1997, ItenL.E et al 1976, Davis et al 1990, Tsonis P.A et al 2004). Interestingly, not only newt possess wider spectrum ofregenerative ability, it is also noted there is no sign of age related declinein their regenerative ability. Newts can regenerate lens, and removing lensrepeatedly for 18 times on the same animal revealed regenerate of lens notaltered by repeated injury.

(Eguchi G et al 2011).   Apart from the appendages, newts also possess ability toregenerate injured CNS, spinal cord and brain. Spinal cord regeneration hasbeen extensively studied in newts. Most of the spinal cord studies have beendone after amputation of the tail. However, transection of spinal cord proximalto the hind limb was performed to study the regenerative ability and behaviourresponse.

In this context, the newts were able to regenerate the spine andrecovered hind limb movement by four weeks after spinal cord injury (Davis etal 1990). Spinal cord injury was also performed to assess the axonalregeneration contribute of cells to regeneration (Zukor et al 2011). Thepaedomorphic salamander, axolotl, has also been extensively studied for theirspinal cord regeneration. These experiments indicate that neural stem cell-mediatedproliferation contribute to spinal cord regeneration (Albors A.D et al 2015).  Adult newts are able to regenerate parts of the brain aftermechanical lesioning. In classical experiments in amphibians the approach wasto remove the optic tectum and study their functional outcome. In newts,removal of optic tectum and assessment of the brain till 90 days indicates thatthey are able to regenerate the optic tectum (Minneli G et al 1987).

In anotherstudy, the retinotectal projection pathway was analysed after partial optictectum removal. This study has revealed that the newts regenerate the optic tectumand most of the retinotectal projection were recovered by 8 months (Okomoto Met al 2007). Recently, number of studies on brain regeneration has beenperformed on newts and this will be discussed later in this chapter.  AS1You need to reference this section


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