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The neuromuscular junction (NMJ), the synapse formed between lower motor neuron and skeletal muscle fibre, is known to be a target in a number of neurodegenerative conditions, including motor neuron disease (MND). Located in an accessible part of the peripheral nervous system, the NMJ can be used as a ‘model synapse’ in the context of ‘connectomics’ – the study of synaptic connectivity throughout the nervous system as a whole. Although the NMJ has been studied in a number of species, relatively little is known about its structure in humans, complicating the translation of animal models of disease to the human condition. Described here is the first detailed cellular and molecular characterization of the human NMJ. A standardized methodology for comparative morphometric analysis of NMJs was developed and validated (‘NMJ-morph’). NMJ-morph was used to generate baseline data for 2160 NMJs from a single litter of wild type mice, representing 9 distinct muscles across 3 body regions. Principal components analysis (PCA) revealed synaptic size and fragmentation to be the key determinants of synaptic variability. Correlation data revealed the pre-synaptic cell (motor neuron) to be a stronger predictor of synaptic morphology than the post-synaptic cell (muscle fibre). Other factors influencing synaptic variability were in a clear hierarchy: muscle identity accounted for more variation in synaptic form than animal identity, with side having no effect. Human tissue was obtained from 20 patients (aged 34 to 92 years) undergoing lower limb amputation, primarily for the complications of peripheral vascular disease (PVD). Muscle samples were harvested from non-pathological regions of the surgical discard tissue. 2860 human NMJs were analyzed from 4 distinct muscles (extensor digitorum longus, soleus, peroneus longus and peroneus brevis), and compared with equivalent NMJs from wild type mice. Human NMJs displayed unique morphological characteristics, including small size, thin axons, rudimentary nerve terminals and distinctive ‘nummular’ endplates, all of which distinguished them from equivalent mouse NMJs. The previous notion of partial occupancy in human NMJs was disproved. As in mice, the pre-synaptic cell was shown to correlate more strongly with NMJ morphology; in contrast to mice, the human NMJ was found to be relatively stable throughout its 90+ year lifespan. In support of the tissue harvesting procedure, patient co-morbidities (diabetes mellitus and vascular disease) did not significantly impact NMJ morphology. Super-resolution imaging of the NMJ revealed significant differences in the functional architecture of human and mouse active zones. Despite the smaller synaptic size in humans, the total quantity of active zone material was conserved between the species, suggesting a homeostatic mechanism to preserve effective neurotransmission. Parallel proteomic profiling demonstrated further species-specific differences in the broader molecular composition of the NMJ. The cellular and molecular anatomy of the human NMJ is fundamentally different to that of other mammalian species. These differences must be taken into account when translating animal models of disease to the human condition.

Background: Language impairment associated with Motor Neurone Disease (MND) has been documented since the late 19th century, yet little is understood about the pervasiveness or nature of these deficits. The common clinical view among healthcare professionals is that communication difficulties can be attributed solely to the motor speech disorder dysarthria. Recent literature raises the possibility of more central processing deficits. Impairments in naming ability and comprehension of complex grammatical constructs have been frequently reported in some patients with MND. However, there is now growing evidence of spelling impairment, which could suggest the contribution of a more phonologically based deficit. In addition, the close relationship between MND and frontotemporal dementia (FTD) raises questions about the connection between the language impairments seen in MND patients and those documented in patients with the primary progressive aphasia (PPA) syndromes associated with FTD. Aims: This thesis examines the nature of speech and language deficits in people with MND and the extent to which expressive communication impairment can occur above and beyond dysarthria. In particular, the study explores: i) to what extent these language impairments can be attributed to deficits in working memory, executive functioning and/or disease severity; ii) what spelling errors can reveal about the integrity of lexical, phonological and orthographic processing; iii) whether similar patterns of impairment can be seen in PPA syndromes; iv) the relationship between language impairment and bulbar onset; and v) the impact these findings have on clinical management of MND patients. Methods: MND patients from across Scotland with changes in speech and/or language were tested using a neuropsychological battery of experimental and standardised tests of naming, spelling, syntactic comprehension, prosody and phonological and orthographical awareness. Patients were also screened for levels of dysarthria, executive functioning and working memory deficits, and results compared to those of matched controls. Findings: As a group, MND participants performed significantly worse than matched controls on measures of naming, spelling, orthographical awareness, grammatical comprehension, affective prosody and verbal fluency, but not working memory. However, based on patterns of individual impairment, of which spelling impairment formed a distinctive marker, the patient group divided into dichotomous subgroups, with 44% of participants categorised as ‘linguistically impaired’, while the remainder displayed little to no impairment. Those participants identified as linguistically impaired did not differ significantly from other MND participants on measures of disease severity, disease duration or dysarthria severity, although significantly more bulbar onset than limb onset participants were linguistically impaired. Spelling error patterns were suggestive of deficits at both a lexical and sublexical level, and were comparable to those reported in PPA literature. These findings suggest that dysarthria may be masking linguistic deficits in almost half of dysarthric MND patients, and highlight the importance of multidimensional assessment of language for effective clinical management.

Apathy is characterised by a lack of motivation towards goal directed behaviour and is a symptom of various neurodegenerative diseases. There are various tools that can be used to assess apathy but a caveat of these is that they usually assess it as a unidimensional concept. Apathy has been recognised to have a multidimensional substructure. The Dimensional Apathy Scale is the only comprehensive measure designed to quantify neurobiologically-based subtypes, called Executive, Emotional and Initiation apathy. The first aim of this study was to explore multidimensional apathy, and its associations with demographic variables, in healthy, community dwelling adults. Secondly, multidimensional apathy was explored in neurodegenerative diseases, specifically Amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). For each disease group, the validity and reliability of both the self rated and carer rated DAS were also determined. Finally, the association between specific apathy subtype impairments and executive dysfunction was explored in ALS patients. Four hundred healthy community dwelling adults, eighty-three ALS patients (seventy-five carers), thirty-four PD patients (thirty carers) and forty-nine AD patients (eighty-nine carers) were recruited for the questionnaire study. In the healthy community dwelling adults, Executive apathy decreased with age, whereas Emotional increased with age. Gender differences were also shown with higher apathy in males on Emotional apathy. There were also employment differences, in that Executive apathy was higher in unemployed individuals compared to those who were employed. Emotional apathy showed difference in type of employment, where full time employed individuals were significantly more apathetic than those employed part time. These findings were taken into account in selecting the appropriate control samples to match our patient groups. In the patient groups, ALS patients were found to be significantly more impaired on the Initiation subscale when compared to controls. Furthermore, Initiation apathy was found to be the most frequent impairment above abnormality cut-off on the carer rated DAS. PD patients were significantly more impaired on Executive and Initiation apathy when compared to controls. These two subscales were most frequently above abnormality cut-off in the carer rated DAS. Finally, AD patients were significantly more impaired on all subscales when compared to controls and, on the carer rated DAS, global impairment over all subscales was most often reported as above abnormality cut-off. Additionally in AD, there was a significant disparity between carer and patient ratings on Executive and Initiation apathy, indicating patients’ impaired awareness. When comparing patient groups, there was a significant difference between carer rated apathy subtype impairments for each patient group. Validity and reliability of the DAS was found to be robust when compared to standard measures of apathy and depression. In the experimental study, a sample of ALS patients (and their carers) and healthy controls (and their informants) were recruited to complete a battery of neuropsychological tests, the DAS, other apathy and depression measures. ALS patients were impaired on tasks of executive functioning when compared to controls. Furthermore, apathy subtype deficits were associated with executive dysfunction in ALS. In conclusion, apathy is a multidimensional concept that manifests in different subtype profiles dependent on neurodegenerative disease. This has further implications for understanding and assessment of cognitive dysfunction and neuropsychiatric symptoms, such as apathy, in ALS and other neurodegenerative disease patient groups.

Motor neurone disease (MND) is an uncommon neurodegenerative disease that is terminal and has an insidious onset. With no known cause or cure, the disease triggers progressive death of motor neurones that causes increasing difficulties with mobility, communication, breathing and nutrition. Most research focuses on the disease process, but little is known of the illness experience from the perspective of those diagnosed with the disease. The aim of this study was to explore what it is like to live with MND and how people with the disease negotiate with others to exercise choice over the way they live. A grounded theory methodology was used to explore the life world of people diagnosed and living with MND. Data were collected via in-depth interviews, their stories and photographs, poems and books participants identified as important and fieldnotes. The textual data were analysed using constant comparative analysis. The majority of participants experienced difficulty with verbal communication. Some invited a third person to interpret their speech and others used assistive technologies such as Lightwriters and computers. Analysis revealed three constructs that, together, told the story of the MND illness experience. First, was the “diagnosis story” that described the devastating process of repeated tests had on the participants, shattering their trust in the competence of the health care system. The second construct revealed the process of living with MND as cyclical and repetitive requiring constant decision-making to adapt to the ongoing changes connected with the disease. The core theme and basic social process of “maintaining personal integrity” evolved as the third construct. This process underpinned and explained participants’ decision-making. Finally a substantive theory was conceptualised as the illness experience: “maintaining personal integrity in the face of ongoing change and adaptation”. This theory illustrates that the basic social process of maintaining personal integrity is central to decision and choice making while living with MND. The findings have implications for people with MND, their carers, health professionals and service providers. Recommendations include improved counselling services for people at the time of diagnosis; the introduction of nurse specialists to support health professionals, people diagnosed with the disease and their families; open, accessible, realistic health and funding policies.

Human pluripotent stem cell (hPSC)-derived neuronal and glial material presents a relatively new opportunity to model human neurophysiology in both health, and disease. Validation of regionally-defined hPSC-derived neurones and glia cultures thus represents the founding blocks of technology that aims to complement existing models. Principally, the relevance of in vitro hPSC-derived material is determined by how representative it is of native material, yet at present the physiology of these cells remains underexplored. Here, electrophysiology and pharmacology are used to functionally assess hPSC-derived excitatory cortical neurones (hECNs), motorneurones (MNs) and oligodendrocyte-lineage cells in the context of regional-specific properties and maturation. These properties are then examined in material derived from hPSCs generated from patients with neurological disorders. This thesis examines of the properties of GABAARs and strychnine-sensitive glycine receptors (GlyRs) in hECNs by assessing their subunit composition, and compares these with studies which have made comparable investigations of rodent tissue where maturation is associated with a shift in GABAA and GlyR compositions. Using pharmacology and RNAseq analysis, GABAAR and GlyRs in hECNs were found to possess receptor populations typical of those reported in the immature cortex. hECNs generated from patients harbouring a mutation to the Disrupted-in-schizophrenia-gene 1 (DISC1), a candidate schizophrenia gene, were then examined. Imbalances in the excitation/inhibition balance are suspected in schizophrenia and, in this regard, the intrinsic excitability properties alongside expression and composition of major neurotransmitter receptors and intracellular chloride concentration were assessed. No obvious differences in excitability or functional expression of AMPARs, GABAARs or NMDARs were observed between case and control derived neurones. Receptor composition and intracellular chloride concentrations were found to be predominantly immature-like, however, AMPAR composition and intracellular chloride concentration were found to be like that of adult cortical neurones. These data are discussed in the context of modelling DISC1-associated pathologies. Thirdly, MNs from hPSCs generated from ALS patients harbouring mutations on the C9ORF72 gene were examined. The hypothesis that increased glutamate-mediated excitoxicity could, in part, be explained by increased expression of Ca2+- permeable AMPARs was examined. The estimated mean single-channel conductance of AMPARs was found to be high in MNs derived from ALS patients, reminiscent of Ca2+-permeable AMPARs and was reversed by gene-editing of the C9ORF72 mutation. Finally, oligodendrocytes generated from ALS patients harbouring TARDBP mutations were examined. Distinctive electrophysiological shifts in oligodendrocytes-lineage cell development are reported. A similar AMPAR phenotype of elevated Ca2+-permeable AMPAR expression was observed in oligodendrocytes derived from two patient hPSC lines and was rescued in an isogenic, gene-edited line, raising the intriguing possibility of convergence in pathophysiologies in the nature of the overlap between cell-type, AMPAR pathology and excitotoxicity in ALS disease progression mechanisms.

Amyotrofisk Lateral Skleros, ALS, är en neurologisk sjukdom vilken leder till att samtliga kroppens muskler förtvinas och dör. Då sjukdomen saknar bot blir all behandling symptomatisk och individuellt anpassad för varje enskild persons behov. I denna systematiska litteraturstudie har det sökts efter olika sätt att stötta denna patientgrupp då syftet att belysa hur vi som personal kan hjälpa och stötta personer med ALS relaterad dysfagi och andningsproblem till en så bra tillvaro som möjligt skulle belysas.Författarna har funnit att omvårdnaden sällan sätts i fokus. Det är istället de lösningar som tar bort symtomet som fått fokus i flertalet av de artiklar som granskats. Att hjälpa dessa personer till trygga och oberoende människor som kan fortsätta att leva istället för som många av artiklarna visade då det gjordes insatser som ledde till att personerna blev mer bundna till sina anhöriga och sina vårdare.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, were found in both sporadic and familial ALS cases. The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca²⁺ dysregulation as a potential mediator of pathology. Ca²⁺ signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca²⁺ stores content and delayed Ca²⁺ release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca²⁺ release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca²⁺ oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca²⁺ signalling. Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying C9orf72 expansions, TDP-43 pathology was not detected. However, preliminary data indicate that C9orf72 MNs present ER Ca²⁺ dysregulation with significantly high intracellular Ca²⁺ concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2. This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca²⁺ homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.

Syfte: Att beskriva närståendes upplevelser av att leva med en person med ALS. Metod: En litteraturöversikt med kvalitativ metod och en induktiv ansats. Resultat: Sjukdomen ALS är ovanlig och de som drabbas av den är i stort behov av omvårdnad. Närstående till en anhörig som har fått diagnosen ALS, upplever att vårdpersonalen har kunskapsbrist angående sjukdomen och vårdandet. De upplever bristande information om sjukdomens förlopp. Egentiden tas ifrån dem, sådant som förr togs förgivet läggs istället åt sidan, då all fokus ligger på den anhöriges omvårdnad. Slutsats: Närstående väljer ofta att vårda den anhöriga i hemmet, trots det snabba sjukdomsförloppet. Vårdandet av den anhöriga leder till känslomässiga påfrestningar hos de närstående, både psykiska och fysiska. / Purpose: To describe next of kin experiences of living with person with ALS. Method: A literature review with qualitative method and an inductive approach. Result: The disease ALS is unusual and those who suffer from it are in great need of nursing. The next of kin those who has been diagnosed with ALS, find that healthcare professionals have a lack of knowledge regarding the disease and care. They experience insufficient information about the course of the disease. The true time is taken away from them, as was previously taking for granted, instead being put aside, as all focus is on the patient's nursing. Conclusion: Next of kin often choose to care for their relatives in spite of the progress of the ilness. The care of the relatives leads to emotional stresses of the related, both mental and physical.