A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva caracterizada pela perda de neurônios motores levando rapidamente os pacientes à morte. Os mecanismos da morte neuronal são desconhecidos e estudos recentes indicam que a microglia pode participar deste processo. Para investigar o papel da microglia na ELA, camundongos transgênicos SOD1G93A foram utilizados na avaliação da morte e do trofismo neuronal em sistemas de co-culturas neurônio/microglia e do efeito do meio condicionado microglial sob os neurônios motores. Ambas as células foram extraídas da medula espinal de camundongos transgênicos (TG) e wild type (WT). A microglia foi obtida dos animais neonatos e adultos na fase pré-clínica da doença. Os neurônios extraídos de neonatos foram marcados com reagente específico para morte neuronal e seus prolongamentos foram quantificados em contraste de fase por métodos estereológicos específicos. A expressão gênica de moléculas candidatas à participação do processo neurodegenerativo relacionadas com a microglia foi quantificada pelo PCR em tempo real, assim como a quantidade de moléculas secretadas no meio condicionado das culturas microgliais dosada pelo ELISA sanduíche. O meio condicionado da microglia TG neonatal não foi capaz de acentuar a morte neuronal, entretanto a neurodegeneração foi potenciada nas análises das co-culturas. Interessantemente, o meio condicionado das microglias WT provenientes de animais adultos foram capazes de aumentar os prolongamentos neuronais feito não observado em relação às microglias TG. Quantidades aumentadas do fator de necrose tumoral ?, interleucina-6 e fator de crescimento derivado do nervo foram quantificadas no meio condicionado das microglias TG. A microglia TG neonatal apresentou dimuição na expressão gênica de AKAP13, HIPK3, UBE2I E NTF5. Esses achados precoces sugerem perda de migração microglial, perda de resistência à apoptose, desbalanço entre proliferação e morte celular e diminuição do suporte trófico neuronal. Em conjunto, os resultados evidenciam a participação da microglia nos mecanismos da ELA / Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disorder affecting motoneurons and leading the patient to death.The cause of motor neurons degeneration in ALS is uncertain and there is any treatment able to prolong the patient life. Recent studies show that microglia could participate of the process of ALS degeneration. Its activation is linked to the disbalance of neuroprotective and neurotoxic action. To investigate the microglia role in ALS, SOD1G93A transgenic mice that develop symptoms similar to the clinical disease were used. We evaluated the neuronal death and trophism in microglia/neuron co-cultures system and in microglial conditioned medium effect under the neurons. Neurons and microglia were extracted from transgenicor wild type mice spinal cord. Microglia was obtained from 1 day pos natal pups and adult onset of disease mice. Cells were stained with a specific marker to neuronal death. Neuronal extensions area and neuronal death was quantified by stereological method. The genic expression of candidate molecules related to degeneration in ALS was quantified by real time PCR and the release of some molecules were quantified by ELISA sandwich. Results showed that maybe transgenic neonatal microglia is not able to increase the neuronal death through releasing molecules in its conditionated medium, on the other hand when transgenic microglia was co-cultured with any kind of neuron, neuronal death was observed. Microglia from adult transgenic mice was not able to promote a neuroprotection compared to wild type in co-culture and conditionated medium experiments. In addition to this, was observed increased tumor necrosis factor alpha, interleukin 6 and nerve growth factor secretion by transgenic microglia. Neonatal transgenic microglia also exhibited reduced genic expression of AKAP13, HIPK3, UBE2I and NTF5. These findings at an early stage suggest a lost of migration potential, lost of apoptosis resistance, disbalance of proliferation and cell death and reduction of neuronal trophic support. So together, these data indicate the involvement of microglia in ALS mechanisms

Motor neuron disease (MND) is a set of neuromuscular diseases that affect the upper and/or lower motor neurons, resulting in progressive disability. Amyotrophic lateral sclerosis (ALS) and Primary lateral sclerosis (PLS) are two forms of MND that both involve upper motor neuron degeneration, which can also accompany extra-motor changes in cognitive, behavioral, and/or emotional functioning for some individuals. Characterization of the cognitive profile of MND is still evolving, with growing interest in cognitive subtypes. The development of cognitive screens targeted to the MND cognitive profile aim to provide efficient and accurate brief assessments. However, empirical evaluation of tailored MND cognitive screens is needed for cross-validation independent of tests’ original developers. The present study addresses the cognitive profile of MND and the utility of brief cognitive screens with a focus on impairments in the language domain. The two primary aims include: (1) comprehensive assessment and characterization of language dysfunction in MND, and (2) empirical evaluation of brief cognitive screens with regard to detecting language impairments. Forty-one patients with MND (ALS n = 36; PLS n = 5) were administered a comprehensive language battery to classify cognitive impairment (MND/ALSci; Strong et al., 2017) in the language domain and/or verbal fluency. Patients also completed two tailored cognitive screens [ALS Cognitive Behavioral Screen (ALS-CBS), Edinburgh Cognitive and Behavioral ALS Screen (ECAS)] and one general screen (Montreal Cognitive Assessment; MoCA). The current preliminary results suggest language dysfunction in MND is characterized by prominent difficulties with word retrieval (confrontation naming) and/or syntax comprehension. However, evidence of reduced word production resembling nonfluent/agrammatic aphasia was not found. In total, 19.5% of the sample met criteria for MND/ALSci in the language domain (n = 8, all ALS); 22.0% met criteria for MND/ALSci in the verbal fluency domain (n = 9). Patients were classified into three subgroups, those with broad language impairments (ALSci-L n = 4, 9.8%), phonemic fluency impairments (MNDci-VF n = 5, 12.2%), or both impairments (ALSci-L+VF n = 4, 9.8%). Results also revealed existing challenges in accurately classifying patients with language dysfunction using brief cognitive screens. The ECAS Language subscore offered limited classification of broad language impairments in the present MND sample (sensitivity 50%, specificity 70%). Among the broader cognitive screens, sensitivities to language impairments were: ALS-CBS (100%), ECAS ALS-Specific Score (75%), and MoCA (71%). Convergent validity was demonstrated between outcomes on the ALS-CBS and ECAS ALS-Specific Score (rФ = .59). Discriminant validity was also demonstrated between outcomes on ALS-CBS compared to the MoCA (rФ = .11). Future research is needed to assess whether language dysfunction reflects a distinct MND cognitive phenotype(s) and potential relationships with disease prognosis. Naming and syntax comprehension may be fruitful language screening targets for future research.

Doctor of Philosophy / Abstract Neurodegenerative disorders are a diverse group of disorders that affect specific subsets of neurons. Motor neuron diseases, neurodegenerative disorders of motor neurons, are seen commonly as sporadic cases and less frequently as familial disease forms. The familial forms show genetic and phenotypic heterogeneity. Clinically motor neuron diseases may be seen as rapidly progressive disorders like amyotrophic lateral sclerosis, ALS or slowly progressive disorders like hereditary motor neuropathies, HMN. The only proven causes for motor neuron diseases are gene mutations that lead to motor neuron degeneration in familial disease forms. Only some of these genes have been identified and have contributed greatly to our understanding of the neurobiology of familial and sporadic disease forms. Identification of additional disease causing genes would help enhance our knowledge of the pathophysiological mechanisms underlying all forms of motor neuron disorders, which would lead to early diagnoses, effective prophylaxis and efficient therapies for these disorders. This study aimed to find gene mutations that cause rapid and slowly progressive familial motor neuron disorders in Australian families and to determine their relevance to sporadic forms of motor neuron disease. The familial forms of ALS show reduced disease penetrance, that is, not all gene mutation carriers manifest the disease. This study examines ALS penetrance in a group of Australian families. The most frequently observed mutations in ALS families are cytosolic superoxide dismutase/SOD1 gene mutations. In a collection of ALS families in our centre, families without the common SOD1 gene mutations were genotyped for other ALS genes and loci and studied using genetic linkage and haplotype analyses. Studies in a large Australian ALS family further confirmed genetic heterogeneity in non-SOD familial ALS, all known autosomal dominant ALS genes and chromosomal loci were excluded as cause of disease in this family. Such families can be studied further to identify additional disease genes and loci mapped in other ALS families. These families represent powerful resources for identification of additional ALS genes. Identifying the pathogenic genes in families with reduced disease penetrance may be more relevant to sporadic forms of disease. dHMN is a chronic neurodegenerative disorder predominantly affecting motor neurons. In a large Australian dHMN family, all the known dHMN genes and chromosomal loci were excluded as cause of disease. A genome wide microsatellite screen was performed in this family and genetic linkage was established to a novel 12.98 Mb locus on chromosome 7q34.2-q36. Candidate genes in this large interval will be screened based on their function and expression profile. Identification of a new dHMN locus provides the basis for future identification of a novel gene involved in motor neuron degeneration. Genes in dHMN have been shown to be pathogenic in ALS and Charcot Marie Tooth syndromes. The new locus for dHMN mapped in this project would lead to identification of a novel dHMN gene, which may elucidate the pathogenesis underlying a wide range of neurodegenerative disorders.

<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.

The prevalence of spasticity after first-ever stroke is approximately 20%, but there are no data on the prevalence of disabling spasticity.The reported prevalence of pain after stroke varies between 19% and 74%, whether pain is associated with spasticity is not known. Until now, there is no health economic analysis of patients with spasticity after stroke. Methods: Two groups of patients were studied. Cohort I was a cross-sectional survey. A representative sample of 140 patients was investigated 1 year after their first-ever stroke. Spasticity was defined as ≥ 1 score on the modified Ashworth scale, disabling spasticity was defined as spasticity having such an impact that intervention, e.g. intensive physiotherapy, orthoses or pharmacological treatment, should be offered. Pain was assesed with the Visual Analogue Scale. All direct costs during one year were identified and converted into Purchasing Power Parities US dollar (PPP$). Cohort II was a prospective cohort study. Forty-nine patients were examined at day 2–10, at one month, and at six months after their first-ever stroke. Assessment and definitions were similar as for cohort I. Results: Spasticity occurs within 1 month and disabling spasticity occur within 6 months. After one year, the prevalence of spasticity was 17% and that of  disabling spasticity 4%. Disabling spasticity was more frequent in the upper extremity. There was an independent effect of severe upper extremity paresis (OR 22, CI 3.9–125) and age below 65 years (OR 9.5, CI 1.5–60). The prevalence of stroke-related pain was 21% after one year. Stroke-related pain was associated with paresis (OR 3.1, 95% CI 1.2–7.7), sensory disturbance (OR 3.1, 95% CI 1.1–8.9) and depression (OR 4.1, 95% CI 1.4–13), but not with spasticity as an independent variable. The majority of the direct costs for one year (78%) were associated with hospitalization, whereas 20% was associated with municipality services. Only 1% of all direct costs were related to primary health care and 1% to medication. The mean (median, inter-quartile range) direct cost for stroke patients with spasticity was PPP$ 84 195 (72 116, 53 707) compared to PPP$ 21 842 (12 385, 17 484) for stroke patients without spasticity (P &lt; 0.001).

Cellular fate, once established, is usually stable for the lifetime of the cell. However, the mechanisms that restrict the developmental potential of differentiated cells are in principle reversible, as demonstrated by the success of animal cloning from a somatic genome through somatic cell nuclear transfer (SCNT). An increased understanding of the molecular determinants of cell fate has also enabled the reprogramming of cell fate using defined transcription factors; recently, these efforts have culminated in the discovery of four genes that convert somatic cells into induced pluripotent stem cells (iPSCs), which resemble embryonic stem cells (ESCs) and can give rise to all the cell types in the body. As a first step toward generating clinically useful iPSCs, we identified a small molecule, RepSox, that potently and simultaneously replaces two of the four exogenous reprogramming factors, Sox2 and cMyc. This activity was mediated by the inhibition of the Transforming Growth Factor-\(\beta\) \((Tgf-\beta)\) signaling pathway in incompletely reprogrammed intermediate cells. By isolating these stable intermediates, we showed that RepSox acts on them to rapidly upregulate the endogenous pluripotency factor, Nanog, allowing full reprogramming to pluripotency in the absence of Sox2. We also explored lineage conversion as an alternative approach for producing a target cell type in a patient-specific manner, without first generating iPSCs. A combination of pro-neural as well as motor neuron-selective factors could convert fibroblasts directly into spinal motor neurons, the cells that control all voluntary movement. The induced motor neurons (iMNs) displayed molecular and functional characteristics of bona fide motor neurons, actuating muscle contraction in vitro and even engrafting in the developing chick spinal cord when transplanted. Importantly, functional iMNs could be produced from fibroblasts of adult patients with the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS). Given the therapeutic value of generating patient-specific cell types on demand, defined-factor reprogramming is likely to serve as an important tool in regenerative medicine. It is hoped that the different approaches presented here can complement existing technologies to facilitate the study and treatment of intractable human disorders.

No description available.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease which results from the degeneration of upper and lower motor neurons in the brainstem, spinal cord and motor cortex. Tragically there is no treatment to prevent ALS. The drug Riluzole acts to delay progression, but only by a month or so in this disease that has a survival length of three to five years. The identification of genes that are mutated in patients with ALS would help devise novel therapeutic strategies as much remains to be discovered about the genetics of ALS. Familial forms of the disease account for only 5-10% of patients. Among these familial cases, about 15-20% are caused by mutations in the zinc/copper superoxide dismutase gene, but the genetic basis of the remaining familial cases and the many sporadic cases continues to be largely unknown. / Altogether, the results presented in this thesis came from the use of several strategies to establish the genetic cause of ALS and the related motor neuron disorders like hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). A concerted and collaborative effort was put forth to identify the gene causative for ALS3 on chromosome 18. In addition, a recently reported locus has been confirmed on chromosome 9p for patients that present both ALS and frontotemporal dementia. The major finding involves the discovery of eight mutations in the TARDBP gene in nine patients with sporadic and familial ALS. Furthermore, a large association study evaluated the role of common polymorphisms in the paraoxonase gene cluster in susceptibility to the development of ALS. In the analysis of upper motor neuron diseases, mutations in a novel gene, KIAA0196, were identified for the HSP locus SPG8 on chromosome 8. Finally, the first locus for PLS was discovered on the p-arm of chromosome 4 following genome scan analysis of a large Quebec family with PLS. / These genetic discoveries all contributed novel advances to the field of motor neuron disorders. As more is elucidated regarding the biochemical function of these the proteins encoded by these genes, a more comprehensive picture of ALS and other motor neuron disorders will hopefully emerge.

Movement is central for life, and all animals depend on accurate regulation of movement for purposeful behavior. There is great diversity of movements, ranging between simple and vital breathing movements to minute and subtle movements of the face used to communicate emotions. Consequently, motor neurons, which are the only route of central nervous system output, are essential for all motor behaviors. To control the many motor behaviors expressed by an animal, motor neurons are exposed to a large number and variety of modulating synaptic inputs and have evolved into subtypes with specific functions. In this thesis, motor neuron subtypes and the synaptic input to motor neurons from Renshaw cells and Ia afferents have been studied. Novel molecular markers that identify subtypes of motor neurons are described. Three markers, Chodl, Calca and ERRβ, have been used to study the degeneration of subtypes of motor neurons in a mouse model of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Another marker, 5-ht1d, has been used to record the electrophysiological character of gamma motor neurons. In mice that lack 5-ht1d, motor neurons develop with reduced proprioceptive input. Remarkably, these mice had fewer foot faults than control animals when challenged to cross a narrow beam suggesting that the amplitude of monosynaptic proprioceptive input to motor neurons is not essential for motor coordination. In a final set of experiments, genetic removal of vesicular transport of neurotransmitter from Renshaw cells suggest that Renshaw cells are not integral for motor circuit function or motor behaviors. However, they are involved in the development of motor circuits in the spinal cord. Together, this thesis provides novel molecular tools for studies of motor neuron subtypes and novel data regarding the development and function of spinal motor circuits.