Amyotrophic Lateral Sclerosis (ALS) is an incurable late onset neurodegenerative disorder characterised by the specific loss of motor neurones (MNs). It has been recently demonstrated that Transactive response DNA-binding protein (TDP-43) is the dominant disease protein in both ALS and a sub-group of frontotemporal lobar degeneration (FTLDTDP). Moreover, the identification of TARDBP mutations in familial ALS confirms a mechanistic link between the observed mis-accumulation of TDP-43 and neurodegeneration but also provides an opportunity to establish an in vitro platform to model these diseases, based on patient-derived induced pluripotent stem cells (iPSCs). This study presents the optimization of an iPSC-based platform to study the consequences of TDP-43 M337V mutation in human functional populations of MNs and astrocytes in isolation as well as in co-culture. To develop this platform, two protocols to differentiate patient-derived iPSCs into functional MNs and astrocytes were first optimized, and the obtained cellular populations were then used to characterize the behaviour of mutant TDP-43 and its effect on the different cell types. This study show that it is possible to use iPSC-based platforms to recapitulate in vitro key aspects of TDP-43 proteinopathies such as MN cell autonomous toxicity and TDP-43 accumulation, but they can also be used to highlight previously unrecognised disease specific mechanisms and to test novel therapeutic approaches. Moreover, by performing co-culture experiments it was possible to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of co-cultured neurons. This iPSC-based platform represents an in vitro model to study both the effect of somatic mutations on isolated patient-specific cultures, but also to investigate cellular autonomy and neurodegeneration in the context of TDP-43 proteinopathies.

Bakgrund: Amyotrofisk lateralskleros (ALS) är en obotlig neurologisk sjukdom, där de motoriska nervcellerna bryts ner och påverkar den viljestyrda muskulaturen. Sjukdomen kan leda till förlamning och andningssvikt, vilket kan debutera inom loppet av tre till fem år. Den psykologiska delen är viktig, då människan övergår till en annan fas i livet och påverkas genom de händelseförlopp som sjukdomen medför. Med en obotlig sjukdom har begreppet lidande en stor närvaro i människans liv. För att kunna hjälpa människor måste det framgå vad det är som skapar lidande. Syfte: Att beskriva hur personer med ALS upplever lidande relaterat till sjukdomen. Metod: En kvalitativ innehållsanalys av fyra patografier. Resultat: Resultatet är baserat på fem kategorier, samt elva underkategorier. Resultatet visar på ett fysiskt lidande, ett socialt lidande, ett existentiellt lidande samt ett lidande relaterat till den maktlöshet sjukdomen medför. Men resultatet visar inte endast en negativ bild kring begreppet utan också en upplevelse av acceptans i slutskedet av livet trots lidandet. Slutsats: Att drabbas av ALS innebar en markant förändring i personernas liv. ALS upplevdes generera ett långt lidandeförlopp med många faser, som påverkade både den fysiska och den psykiska hälsan. Lidandet övergick med tiden till en acceptans över situationen. Vidare forskning krävs för att beskriva upplevelser som kan förbättra omvårdnaden. / Background: Amyotrophic lateral sclerosis (ALS) is a non-curable neurological disease, in which the motor neuron cells breaks down and affects the involuntary musculature. The disease can lead to paralysis and respiratory failure, which can debut in the course of three to five years. The psychological part is important, because people enters another phase in life and are influenced by events caused by the disease. With an incurable disease, the concept of suffering is a major presence in human life. In order to help people it must be shown what it is that creates suffering. Aim: To describe how people with ALS experience suffering related to the disease. Method: A content analysis of four pathographies. Results: The result is based on five categories and eleven subcategories. The result shows a physical suffering, a social suffering, an existential suffering and the suffering associated with the impotence of the disease. But the results show not only a negative image about the concept, but also a sense of acceptance in the final stages of life despite the suffering. Conclusion: To suffer from ALS has had a significant change in people’s life. ALS generates a long suffering process with many stages where ALS affected both the physical and mental health. Suffering turned over time into an acceptance of the situation. Further research is needed to describe experiences that can improve the care.

The causes of Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease that results in skeletal muscle paralysis, remain unclear. However, a nuclear DNA and RNA binding protein called TAR DNA binding protein 43 (TDP-43) has emerged as a critical marker of ALS pathology. A previous drug screen conducted in the Zarnescu laboratory showed that anti-diabetic drugs can rescue lethality in a fruit fly model of ALS based on TDP-43. These results suggested that the insulin signaling pathway might be altered in motor neurons in a TDP-43 dependent manner. Therefore, we hypothesized that the insulin pathway is interacting with TDP-43 in vivo and may be contributing to TDP-43neurotoxicity. Using genetic interaction approaches in flies we found that TDP-43dependent locomotor defects are sensitive to the levels of insulin receptor activity. In addition, genetic interaction data suggest that Akt is hyperactivated in motor neurons expressing TDP-43, possibly as a compensatory mechanism to enable survival. Finally, upregulating protein synthesis through S6K and 4EBP appears to have beneficial effects. These findings support our hypothesis and provide insights into potential therapeutic strategies to help treat this devastating disease.

The overall aim of this thesis was to investigate quality of life, coping and emotional distress (i.e. anxiety and depression) among newly diagnosed ALS patients. An additional aim was also to investigate relatives’ experiences of the care for the patient and the support they received for themselves during the disease progression. The most nominated areas of importance for the patient’s overall QoL were family, friends and own physical health. Most patients rated their QoL as good, which did not change at subsequent measurement, despite their physical function having changed for the worse during disease progression. Some patients had symptoms of clinical anxiety and depression during the first year after diagnosis. The total quality of life score did not correlate with physical function but with depression early on after diagnosis. Most patients used support and independence as strategies to cope with the disease during the first six months after diagnosis. There were few changes early on after the diagnosis, and the patients used several different strategies. The results show that the use of coping strategies remained stable over time. Both physical function and emotional distress correlated significant with different coping strategies, with some variation during the disease progression. Relatives experienced the care of their loved one as positive and based on the patient’s needs and desires. The treatment, knowledge, support and help from the staff were important for the relatives’ feeling of security. Different factors influence the use of support for themselves. The relatives did not think of their own needs, but their focus was rather on the patient. The results of the thesis highlight the importance of providing support both to patients and their relatives during the disease progression. With early and regular evaluation on quality of life, coping and emotional well-being among the patients, the health professionals may be able to support the patients based on their specific needs, which probably will increase their quality of life.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

Amyotrofisk lateral skleros (ALS) är ett samlingsnamn för olika neurologiska och obotliga sjukdomar som kännetecknas av nedbrytning av det motoriska nervsystemet. Symtom som svaghet i muskler uppkommer succesivt och orsakar andningssvikt och förlamning. Idag saknas adekvat bromsmedicin mot ALS vilket gör att fokus läggs på symtomlindring. För att kunna ge en god omvårdnad till patienter med ALS är det viktigt med ökad kunskap och förståelse inom hälso- och sjukvården, vilket kan leda till en bättre livskvalitet. Syftet med studien var att undersöka hur patienter med ALS upplever omvårdnaden under sjukdomstiden. Metoden som användes var en litteraturstudie baserad på 11 vetenskapliga artiklar. I resultatet framträdde tre kategorier; att vara medskapare i sin vård som innebar att patienter upplevde att beslutsfattande, kontroll och bristande kunskap påverkade omvårdnaden. Att få individanpassad vård innebar att få stöd, tid och ett gott bemötande. Patienter som upplevde sig bli beroende av omvårdnaden kände sig som en börda och maktlösa. Det är angeläget med forskning som undersöker hur patienter med ALS upplever omvårdnaden eftersom nuvarande forskning främst är inriktad på hur patienter upplever sjukdomen. / Amyotrophic lateral sclerosis (ALS) is a generic name for various neurological and incurable diseases characterized by the breakdown of the motor nervous system. Symptoms such as muscle weakness occur gradually and causes respiratory failure and paralysis. Today there is no cure for ALS, but instead are the sight on symptom relief. In order to provide good care to patients with ALS, it is important to increase knowledge and understanding of health care, which can lead to a better quality of life. The purpose of this study was to examine how patients with ALS experience nursing care during illness. The method used was a literature review based on 11 scientific articles. The result appeared in three categories; to be co-creators in their care which involved patients felt that decision making, control and lack of knowledge affected the care. To get individualized care meant to get support, time and good treatment. Patients felt that they become dependent on nursing felt as a burden and powerless. It is important with research that examines how patients with ALS experience care as current research mainly focuses on how patients experience the disease.

Bakgrund: Amyotrofisk lateralskleros (ALS) är en obotlig sjukdom som drabbar de övre och nedre motorneuronen. Den förväntade livslängden är tre till fem år efter symptomdebut och personen drabbas av succesiva funktionsnedsättningar. Behoven hos personer med ALS förändras hela tiden och därför är det viktigt att omvårdnadsinsatserna anpassas till individen. Syfte: Att belysa personers upplevelser av att leva med amyotrofisk lateralskleros och hur de hanterar sjukdomen. Metod: Allmän litteraturstudie av tio kvalitativa vetenskapliga artiklar. Resultat: Den fysiska förlusten upplevdes skrämmande och det var frustrerande att inte kunna påverka sin situation. Det ledde också till att personerna kände sig som en börda. Det var dock betydelsefullt att trots sjukdomen fortsätta leva ett så vanligt liv som möjligt. Osäkerheten kring framtiden skapade ett behov av att leva i nuet. Resultatet visade även att närstående inverkade positivt på hanteringen av sjukdomen, likaså att acceptera sin situation. Att ha en känsla av kontroll främjade hoppet. Stödet från sjukvården och även att känna sig värdefull ansågs också ha betydelse för hanteringen. Slutsats: Trots att personer med ALS påverkas psykiskt och fysiskt av sjukdom kan de bevara ett vanligt och aktivt vardagsliv genom användning av olika copingstrategier. Klinisk betydelse: Vårdpersonal behöver ha kunskap om hur denna patientgrupp upplever och hanterar sin sjukdom för att kunna förstå, bemöta och vårda dem på rätt sätt. / Background: Amyotrophic lateral sclerosis (ALS) is an incurable disease which affects upper and lower motor neurones. Life expectancy is between three to five years after symptom onset and the person is affected by progressive impairments. The needs of people with ALS are constantly changing and therefore it’s important that the nursing care is adapted to each individual. Aim: To illuminate people’s experiences of living with amyotrophic lateral sclerosis and how they manage the disease. Method: A literature review of ten qualitative research articles. Result: The physical loss felt scary and it was frustrating not to be able to influence the situation. It also led to the feeling of being a burden. It was important though to be able to continue living as ordinary as possible despite the disease. Insecurity concerning the future led to a need of living in the present. The results showed that relatives had a positive impact on the management of the disease, as well as acceptance of the situation. To have a feeling of control promoted hope. Support from health care and the feeling of being valuable was also considered to be important for the management. Conclusion: Even though people with ALS are affected mentally and physically they are able to preserve an ordinary and active everyday life with the help of different coping strategies. Clinical implications: Health care professionals need to have knowledge of how these patients experience and manage their disease in order to understand, respond and care for them in the right way.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the commonest motor neuron diseases of adult- and childhood onset. Alterations of the RNA binding protein TDP-43 are associated with most cases of ALS, while SMA is caused by deletion of the Survival Motor Neuron (SMN1) gene. SMN has been well characterised in its role in the assembly of the cellular machinery that carries out splicing of pre-mRNA, but is thought to have other functions in RNA metabolism unrelated to pre-mRNA splicing. It is conceivable that specific aspects of RNA handling are disrupted in both SMA and ALS. A variety of genetic, molecular and neuropathological approaches were applied to investigate a potential common pathway in these diseases. The spectrum of genetic mutations underlying motor neuron disorders were explored by screening patient DNA. Cell culture and mouse models were used to test the hypothesis that altered pre-mRNA splicing causes motor neuron death. Human neuropathological specimens were examined for changes in proteins involved in RNA metabolism. The results indicate that altered pre-mRNA splicing is a late occurrence in disease and more likely to be a consequence rather than the cause of motor neuron degeneration. However, the notion that RNA metabolism is highly relevant to motor neuron diseases was strengthened by the discovery of mutations in another RNA binding protein, FUS, in cases of ALS without TDP-43 pathology. Overall the findings highlight the need to consider disruption of mRNA transport and regulation of mRNA translation in future motor neuron disease research.

MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB­ C5, expressing OriP sequences of the Epstein­Barr and five reprogramming transgenes Oct4, Sox2, Klf4, c­Myc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject

La Sclérose Latérale Amyotrophique se caractérise par la perte sélective de motoneurones (MNs) du cortex, du tronc cérébral et de la moelle épinière. Les souris surexprimant le gène humain muté codant pour la superoxide dismutase 1 (mSOD1) constitue un bon modèle d'étude. Les MNs mSOD1 présentent une hypersensibilité à la mort après activation du récepteur Fas et la production d'oxyde nitrique (NO). Notre étude protéomique a identifié deux effecteurs du NO, la calréticuline (CRT) et CRMP4. CRT est une protéine chaperonne de stockage du calcium dans le réticulum endoplasmique. Nous montrons que, in vivo, CRT diminue de moitié dans une sous population de MNs mSOD1 dits vulnérables, car dégénérant les premiers. Sa diminution est nécessaire et suffisante pour induire la mort des MNs mSOD1 en activant le stress du RE. CRMP4 est une protéine de régulation de la croissance axonale, qui augmente in vivo dans les MNs mSOD1 à un stade pré-symptomatique. Sa surexpression est suffisante pour induire une dénervation périphérique et la dégénérescence de MNs mSOD1. Nos résultats mettent en évidence CRT et CRMP4 comme étant deux cibles thérapeutiques potentielles dans la SLA. / Amyotrophic Lateral Sclerosis (ALS) is characterized by the selective degeneration of upper and lower motoneurons (MNs). The most common familial form and best characterized mouse model of ALS is linked to mutations in the gene coding for the superoxide dismutase 1 (mSOD1). MNs expressing mSOD1 show an increased sensitivity to the death induced by Fas/NO activation. Our proteomic study identified two downstream effectors of NO, Calreticulin (CRT) and CRMP4. CRT is a chaperone-calcium-binding protein of the endoplasmic reticulum, which is decreased two-fold in vivo, in an early degenerating MNs sub-population, named vulnerable. The decrease in CRT expression is both necessary and sufficient to kill mSOD1 MNs through ER stress activation. CRMP4 is a neurite outgrowth regulator which expression is increased in vivo in mSOD1 MNs at a presymptomatic stage. CRMP4 overexpression is sufficient to induce peripheral denervation and, through a dying-back effect, to kill mSOD1 MNs. Our results point out CRT and CRMP-4 as two potential therapeutic targets for ALS.