AbstractAmyotrophic substantial cognitive and behavioural phenotype (ALS-FTD).Mutation in

AbstractAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects the motor neurones in the brain, brainstem and the spinal cord. ALS, first described by the neurologist Jean-Martin Charcot takes this name by the degeneration of upper motor neurones (UMNs) and its descending axons from the motor cortex to the lateral portion of the spinal cord (lateral sclerosis); and the demise of lower motor neurones (LMNs) with denervation and muscle atrophy (amyotrophic). Classically, ALS has been described as pure motor neurone disease although recently, cognitive and behavioural abnormalities have been found in patients.  Now, ALS is recognized as a motor neurone disease with substantial cognitive and behavioural phenotype (ALS-FTD).Mutation in the gene FUS accounts for approximately ~5% and ~1% of familial and sporadic ALS cases, respectively.

Patients carrying FUS mutations present an aggressive and juvenile onset contrary to mutations in other ALS genes. FUS inclusions observed in surviving motor neurones and frequently in oligodendrocytes may be positive to ubiquitin or P62, demonstrating alteration in several cellular pathways. We are using the FUS?14 mice, which carry a de novo mutation leading to a truncated FUS protein lacking the nuclear localization signal  (NLS). This mutation is based on a 22-years old patient having an aggressive, juvenile sporadic ALS onset. Our experiments aim to further characterize the initial pathological and molecular events leading to ALS-like phenotype in our FUS? mice. phenotype the motor cortex pathology and identified the starting timepoint of NMJ denervation using the FUS?14 mice.

 We are exploring the muscle fibertyping and the metabolic shift to fully characterize the muscle phenotype.CHAPTER 1: INTRODUCTION1. Amyotrophic Lateral Sclerosis (ALS)1.1. Clinical features of ALS1.2. Epidemiology of ALS1.3.

Genetics of ALS1.3.1. Familial ALS1.

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3.2. Sporadic ALS1.3.3. Genes causing ALS (ALS genes)1.4. Risk factors for ALS1.

4.1. Age1.4.

2. Genetics1.4.3.

Environmental 1.5. Neuropathology of ALS1.5.1. Classic neuropathology  1.5.

2. Molecular neuropathology1.5.3. Involvement of non-neuronal cells 1.6. FUS-ALS mice models 1.6.

1. Transgenic FUS-ALS mice models1.6.

2. Gene editing FUS-ALS mice models  1.6.1. FUS?14 mice model1.7 Research aims using the FUS?14 mice1.

1. Clinical features of ALSJean-Martin Charcot (Paris, 1825-1893) correlated the anatomy with the clinic (anatomoclinical method) to devise the most evident pathological features of ALS  (). Charcot and collaborators firstly described that chronic progressive paralyses and contractures are linked to lesions in the lateral column of the spinal cord but patients did not have muscle atrophy  (). Secondly, they also found that lesions in the anterior horn of the spinal cord provoke paralysis and extensive muscle atrophy without contractures . Taken their observations together, they concluded that 1) there is a connection between the anterior side of spinal cord and skeletal muscle, 2) lesions in adjacent but anatomical different regions in the spinal cord result in a different clinical presentation and 3) that spinal cord and skeletal muscle are integral components of the motor system  (). The anatomoclinical method used by Charcot, although incipient, is still considered valid as it clearly shows the specific features  of ALS  ().

 ALS clinical features are not observed as single set of symptoms but as heterogeneous manifestation with motor neurone degeneration  (). In around 66% of ALS patients, clinical symptoms may start focally in the limbs, often in the arms, and then spread to other regions  (). First clinical signs in these patients may begin with difficulty in lifting the frontal side of the foot (foot drop) or losing hand dexterity  (). Arms or leg weakness accompanied with spasticity is also common  (). When clinical symptoms worsen the patient´s health, gait and then competence to walk are loss progressively  (). Ultimately, patients die of respiratory failure within 3-5 years after initial diagnosis  ().

ALS initial symptoms can be divided into bulbar- and limb-onset ALS. Early symptoms may begin in the arms or legs, being referred as limb-onset ALS  (). When symptoms appear as difficulty in speech or swallowing, it is termed bulbar-onset ALS  ().1.2. Epidemiology of ALS           Similar to most neurodegenerative diseases, ALS is a rare or orphan disease with a worldwide prevalence of about 2-3 cases per 100,000 people older than 15 years old, although most of this data come from epidemiological base-studies conducted in European population . The incidence of ALS in men is slighter higher than in women (1:350 vs 1:400 respectively) probably due to higher rates of spinal onset ALS among males .

ALS frequency appears to be slightly variable between European-derived groups. For other ethnic groups, if enough data are available, the risk to develop ALS seems to be slightly lower than the European´s extraction (). For example, Finland has the highest ALS incidence (2.4-6 per 100000 inhabitants) while Italy; a southern European country with higher admixture has an incidence of 0.6 per 100000 inhabitants  (). In Uruguay, a population of mostly European background, ALS rates remains to those found in southern European countries such as Spain, Portugal and Italy  ().

In those populations with higher levels of admixture like USA, Hong Kong and Cuba, ALS rates appears to be lower than the Europeans one  (). However, if this is true, there are insufficient data from other vast areas of the world- including some east European countries with mostly white population, large areas of Asia, Africa and South America- to confirm this hypothesis  (). Overall, the world ALS incidence and prevalence are ~ 2 and ~ 5 per 100000 per inhabitants each year, respectively  ().1.

3. Genetics of ALS1.3.1. Familial ALS About 5-10% of ALS cases are caused by gene mutation and, thus, they are transmitted in families  ().

These cases are named familial ALS (fALS). The remaining 90% of ALS cases are considered sporadic (sALS ). It is sufficient that two members of a family have the same mutation in an ALS gene to be considered as fALS, however, if no possible to demonstrate this due to death of one of the relative or when live far away, this may be considered as sALS. To demonstrate how arbitrary the definition of fALS and sALS is, familial clustering -same gene mutations in related individuals- may happen stochastically without a genetic basis  ().Most of the mutations in fALS are missense substitutions but transmitted as dominant and with relative high penetrance . Several genes have been strongly associated with ALS and they can be grouped into few categories (table 1): genes involved in protein quality, genes involved in RNA metabolism and stability, genes participating in cytoskeletal dynamics.


Sporadic ALSThose cases with de novo mutation (sporadic) or that cannot be explained with genetic background are considered sporadic ALS  ().Table 1. Genes causing ALS in Humans. Taken from J.P. Taylor et al 2016 ().1.

3.3. Genes causing ALS (ALS genes)SOD1 Mutations causing ALS has been reported since 1993 (), where SOD1 was the first gene to be recognised as ALS causative  (). More than 150 mutations have been found in the SOD1 gene ever since although it remains unclear the mechanisms causing ALS  (). SOD1-ALS patients have a variable phenotype ranging from the most aggressive clinical presentation (SOD1 A4V mutation) leading to death in 12 months  () to a slowly disturbing effect in the patients carrying SOD1 D90A mutation  (). SOD1-D90A patients developed respiratory failures with little cognitive impairment after 10 years of initial diagnosis  (). SOD1 mutations cause about 20% of fALS and 2% of sALS cases  () and surviving motor neurones contain SOD1 positive inclusion and Bunina Bodies.TARDBPAnother important contributor to genetic of ALS is TARDBP gene.

This gene explains more that 5% of fALS and less than 1% of sALS  (). Strikingly, TARDBP-codifying protein TDP43 is the major component of ubiquitin inclusions in surviving motor neurones  in ALS and FTD cases  (). Most mutations are located near the C-terminal region of TDP43 leading to splicing and ribonucleoprotein binding alterations  ().FUS2Fused in sarcoma gene (also known as translocated in Liposarcoma or TLS gene) codifies the FUS protein, a ribonucleoprotein member of FET family  (). More than 40 mutations have been found in the FUS gene since 2009 where it was discovered that FUS mutation causes ALS  (). Although mutations in the FUS gene causes a small number of fALS and sALS (~5% vs less that 1%), FUS protein shares many functions with TDP43 protein, suggesting that alteration in RNA metabolism is an important event leading to ALS  ().

Mutation in the gene FUS have an aggressive and short disease onset, usually less than 24 months ().FUS protein is an RNA binding protein involved in all aspects of RNA metabolism (). Most mutations cluster in the C-terminal region (figure 1.2) where the non-canonical nuclear localization signal is located (PY-NLS ) ().

Thus, FUS accumulates in the cytoplasm due to loss of PY-NLS leading to cellular stress  (). Surviving motor neurones in this ALS subtype, also known as ALS6, have positive FUS inclusions but TDP43 negative . These motor neurones also contain ubiquitin or P62 inclusions, basophilic inclusions (BI) with a wide range of shapes (blue-grey colour by Hematoxylin&Eosin ) (figure 1.3 ). Figure 1.2.

Schematic of FUS protein and its mutation. Taken from Deng H. et al., 2014 ().Figure 1.3. Motor neurones inclusions in ALS pathology. A) Basophilic inclusions by H&E staining (BIs).

B) Motor neurones containing round inclusions as observed in A. C) FUS inclusions observed by immunohistochemistry (IHC). D) Glial inclusions in ALS brain observed by IHC. Taken from Mackenzie I.R.

A. et al., 2017.1.4.

Risks factor for ALS           1.4.1.

AgeAs observed in many neurodegenerative disorders like Parkinson´s disease (PD) and Alzheimer’s disease (AD), age is undoubtedly a risk factor for ALS    (). The risk to develop ALS increases with age but elder population are more likely to be located in non-neurological consultancy, as some initial symptoms of ALS may be considered typical of this age group  (). Thus, real ALS frequency in old patients (> 65 years old) may be difficult to determine   (). 1.4.2.

GeneticsALS inheritance pattern depends on the gene mutation but it usually follows a Mendelian pattern of transmission with high penetrance particularly in familial ALS  (). Regretfully, these genes only explain a small portion of ALS cases. Classically, genetic causes of ALS are explained when one or more members of a family carry a mutation in an ALS gene. This scenario is enough to classify this case as familial ALS, but familial clustering may happen without genetic evidence or by chance  (). This apparent contradiction may lead to underestimate the number of fALS.                       1.4.

3. Environmental risk factor for ALSEnvironmental risk factors for ALS have been assumed but not fully established. Smoking may be a risk factor among post-menopausal women but not among men  (). It has been suggested that higher intake of vitamin E is associated with lower risk of ALS  (). In different studies, individuals having strong physical activities (American footballer, soccer and rugby players) were suggested to have higher risk for ALS due to repetitive head injuries, intake of drugs to treat injuries ( ). Workers in a wide range of occupational activities (construction and electrical workers, house painters and power production plant workers, among others) have been associated with a higher ALS risk but again; no definitive conclusions have been established (). On the other hand, the atypical aminoacid ?-methylamino-L-alanine (BMAA) produced by the cyanobacteria  () is thought to cause the high incidence of ALS in the pacific island of Guam  (ALS-Guam) and Kii Peninsula although currently, ALS incidence and prevalence in those areas dropped to average values  ().1.

5. Neuropathology of ALS1.5.1. Classic neuropathological features of ALS   Jean-Martin Charcot first made the clinical and histopathological correlation of ALS in the 1860´s ( ). Macroscopically, brain of patients with ALS does not have gross abnormalities except for those overlapping with frontotemporal dementia (FTD) where atrophy of the frontal and/or temporal cortex is more evident ( ).

The spinal cord exhibits loss of myelinated axons in the lateral and anterior column s (). Additionally, reduction in the white matter of the corticospinal tract is frequently observed  (). Microscopically, the brain and spinal cord of ALS patient undergo a severe atrophy. Vladimir Betz and Bunina T.

Zhurnal reported the loss of pyramidal cells (also known as Betz cells) and eosinophilic inclusions (also known as Bunina bodies) in 1874 and 1961, respectively ( ). Bunina bodies, observed in both sALS and fALS, are mostly observed in cytoplasm of motor neurones in the spinal cord and brainstem, but it can be rarely found in Betz cells, Oculocomotor and Onuf´s nuclei  (). Ultrastructurally, Bunina bodies contain an amorphous electron-dense substance with various central areas having cytoplasmic elements. Bunina bodies’ role in ALS remains unknown  ().1.5.2. Molecular neuropathology of ALSIn the past few decades, the discovery that surviving motor neurones containing cytoplasmic protein aggregation in post-mortem ALS tissue has driven to the better understanding of ALS pathology.

The first to be discovered was ubiquitin inclusions in 1988 , a protein involved in recognizing proteins to be processed by the ubiquitin-proteasome system  () (). These ubiquitin inclusions are mostly found in the cytoplasm of lower motor neurones, occasionally in the glial cells and rarely in temporal cortex, hippocampus and striatum  (). Depending of their morphology they can be named as: skein-like or round ubiquitin positive inclusions .

  P62, a protein ultimately involved in phagocytosis is also found in surviving motor neurones. More recently, other proteins have been found to aggregate or to be phosphorylated in surviving motor neurones. Those proteins include FUS, TDP43, SOD1 and protein product of C9orf72 gene among others  (). INCREMENTAR ALGO…1.5.3. Involvement of non-neuronal cells in ALSAstrocytesBrain inflammation mediated by glial cells has been recognized as an important contributor to the pathology of ALS  ().

Dying motor neurones in both human ALS patients and ALS-animal model are surrounded by reactive astrocytes  (). These reactive astrocytes exhibit greater expression of glial fibrillary acidic protein (GFAP), a type III intermediate filament involved in cell communication and proper function of Blood-brain barrier (BBB) in the central nervous system (CNS ) (). Higher expression of GFAP in astrocytes is normally observed in the grey matter in the ventral horn of the spinal cord in ALS patients  ().  Reactive astrocytes also expressed S100? and inflammatory markers such as inducible nitric oxide synthase (iNOS) and neuronal NOS ().Microglia Microglia is a type of macrophage cell resident in the central nervous system that, unlike the other cells in the brain, embryologically derived from mesodermal tissue  (). This cell type is normally observed in its ramified shape in healthy brain and spinal cord  ().

Microglia are particularly sensitive to any insult and play a double-sword function: it protects against inflammation and when in its reactive state, it releases a number of pro- and anti-inflammatory molecules that may increase the inflammation state in the brain and the spinal cord  (). In brains of ALS patients, activated and phagocytic microglia release large number of pro-inflammatory molecules such as cytokines (tumour necrosis factor (TNF- ?) and interleukin 1?), chemokines (monocytes chemoattractant protein-1 (MCP-1) and macrophage colony stimulating factor (MCSF)), and neurotrophic factors such as insulin-like growth factor-1 (IGF-1), among others  (). Furthermore, activation of microglia has been correlated with severity of upper motor neurone degeneration in human ALS   (). Myelin-producing cellsOligodendrocytes are another glial cell in the central nervous system but until recently its role in ALS has been poorly discussed  ().

Oligodendrocytes produce myelin and provide insulation to axons in the brain and those extending to the spinal cord  (). In post-mortem human ALS brain tissue, diffuse myelin in the lateral columns of the spinal cord is observed together with reduction in the numbers of small fibres  (). Schwann cells, equivalent of Schwann cells in peripheral nervous system (PNS), synthetize myelin in the PNS and participate in the development and regeneration of the axon  ().1.6. FUS-ALS mice models Since the discovery that SOD1 gene causes fALS in 1993, many other genes have been identified as ALS causative  (). Following SOD1, TARDBP, FUS/TLS, C9orf72, VCP among others have been identified and different mice models have been developed using either transgenic or gene targeting technology.

1.6.1. Transgenic FUS-ALS mice models1.

6.2. Gene editing FUS-ALS mice models 1.

7.1. FUS?14 mice modelThe FUS?14 mice was developed using gene-targeted technology based on a report of a 20-years old female patient with frameshift ALS mutation (FUS p.G466VfsX14 )(). This heterozygous mice carries an endogenous copy of a mutated FUS gene based on a 22-years old patient carrying a fameshift mutation  (). This mutation is a point subtitution (A>G) in the splice aceptor site of the exon 14 leading to FUS protein lacking the NLS region (figure 1.4 ).  Figure 1.

4. Schematic of the mice FUS gene showing the splice site mutation introduced.The FUS?14 mice carries one copy of wildtype FUS and truncated FUS protein. Protein level of truncated FUS in the FUS?14 mice does not differ signitifcantly from the wildtype littermate  (). A lifetime analysis at 22 months of age showed that our mice model had a significant reduction in survival time compared to wildtype littermates  (). The FUS?14 mice does not have motor impariment at 3 or 6 months of age and no evident ALS phenotype is observed according to Locotronic and gait analysis  . However, at 12 months of age, the FUS?14 mice displays a significative increase in the hind-limb errors but not in the fore-limbs  ().

This phenotype worsen at 15 months of age suggesting that 1)motor phenotype is more evident in older mice and 2) more suceptibility in hind-limb muscle to truncated FUS protein .  At 18 months of age, FUS?14 mice had a significative gait alteration when compare to wildtype littermate  ().They carried out a longitudinal analysis of motor neurones in the ventral horn of the spinal cord at 3, 12 and 18 months of age.

As observed before, they found not difference in lower motor neurones counting at 3 months of age, but a reduction of them at 12 (-14%) and 18 months of age (-20%). Similarly, intact end-plate innervation of the NMJ was reduced in the FUS?14 mice (58%) when compared to wildtype mice (85 %).Figure 1.5. Longitudinal  motor neurones counting in the FUS?14 mice.

As truncated FUS in our heterozygous mice lacks the PY-NLS motif, it is expected to accumulate in the cytoplasm. However, our mutant FUS protein is not completed depleted from nucleus. Importantly, we did not observe aggregation or insoluble mutant FUS but some accumulation at the perinuclear site was observed. No ubiquitin or P62 pathology was observed  (). Taken together, these results suggest that a missense mutation in the endogenous FUS gene causes no developmental effect in the mice, but an ALS-like phenotype (figure 1.6). This mice model undergoes a pathological shift before 12 months of age thus we aim to use molecular techniques around this time point.Figure 1.

6. Schematic of FUS?14 mice phenotye through different time points.1.

7 Research aims using the FUS?14 mice1.7.1. Objectives of the PhD thesis Using the FUS? mice we have demonstrated progressive lower motor neurones death and motor dysfunction starting at 12 months of age and worsening at 18 months of age  in this model (). Following with the further characterization of this mice model, we will focus first on studying the brain pathology, and second on the NMJs and muscle physiology. General objective: My PhD thesis aims to characterize the initial pathological and molecular events leading to ALS-like phenotype in our FUS? mice. Objective first: study of brain pathologyAs explained above, ALS is defined as degeneration of both upper and lower motor neurones thus we will explore whether motor neurones in motor cortex of FUS? mice die and if associated with brain gliosis and protein pathology (ubiquitin and P62 aggregation). We will also explore the brainstem nuclei (upper motor neurones) as the patient carrying the mutation used to develop our animal model had bulbar onset ALS with LMN signs.

 Objective second: study of the NMJ and muscle pathologyAccording to the dying-back hypothesis, the motor axon terminal at the NMJ level is thought to be the initial place where pathological changes start and then it travels retrogradely leading to ALS. We will also focus on muscle fibertyping and its metabolism as this has been demonstrated to contribute to muscle atrophy.Third objective: Molecular studies of NMJsWe have observed NMJ denervation of 18 months and progressive motor neurone degeneration and motor phenotype starting at 12 months of age in the FUS? mice, suggesting that “early molecular and pathological changes” may start before the phenotype is evident. With this in mind, we aim to explore the molecular mechanism leading to NMJ denervation and whether the distribution of the truncated FUS in the axon terminal is disrupted in our mice model.

 1.7.2. Importance of the FUS?14 miceThe FUS?14 model is novel, carries a truncated mouse FUS expressed at endogenous levels and shows a progressive ALS-like phenotype  ().

This is the first mice model to have an identical point mutation  (A>G) in the exon 14 of the endogenous gene FUS leading to a frameshift FUS mutation to exactly mimic the mutation of the patient used to develop this mice model  (). And together with the FUS model developed by Scekic-Zahirovic et al. 2016., where was done using homologous recombination, these are the only two mice models using endogenous FUS locus expressing FUS protein at physiological levels. The FUS? mice model is also an important tool to study the FUS biology and the toxic-gain of function, a mechanism proposed to explain motor neurone death. Most importantly, this mice model has a progressive but slow ALS-like phenotype as there is not evidence of motor system alteration at 3 months but death of motor neurones at 12 months of age. Thus we can investigate the earliest pathological changes and FUS dysfunction in a heterozygous mouse. Aunmenta algo


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